Journal of the Endocrine Society

Online ISSN: 2472-1972
As a part of our endocrine fellowship quality improvement training, we sought to improve patient access to endocrine outpatient clinic appointments at our institution. From the physician perspective, patients who do not show up for a 30-45 minute new patient visit slot disrupt clinic flow and limits access for other patients. Therefore, we decided to focus our attention on reducing the new patient no-show rate. Here, we assessed the efficacy of our current scheduling system from 2014-2016. This analysis was performed as part of a quality improvement project to inform future interventions. Previously, only a few studies have attempted to identify baseline no-show rates (1,2). Our institution utilizes an automated telephone appointment reminder system (Televox) which calls patients three to four business days prior to a scheduled appointment and gives the patient an opportunity to confirm or cancel the appointment. Televox data for 7,556 patients over 34 months (1/2014-10/2016) revealed a 95% show frequency for patients who confirmed their appointment, 82% show frequency for patients for which there was no response to the Televox reminder, and 78% show frequency of patients who were not reached (Televox call not made). P-Chart of the show frequencies of patients who did not respond or were not reached fluctuated between 70-90% over this period. This analysis shows that new patients who did not respond or were not reached are a potential target for intervention. By identifying this cohort early, we will be able to implement intervention and reschedule patients in a timely manner to improve clinic flow and clinic access. References: 1. Mohamed, K., Mustafa, A., Tahtamouni, S., Taha, E., Hassan, R. A Quality Improvement Project to Reduce the ‘No Show’ Rate in a Paediatric Neurology Clinic. BMJ Quality Improvement Reports. pii: u209266.w3789, 2016. PMID: 27651897 2. Santiago, V.A., Warwick, K., Ratnakumarasuriyar, S., Oyewumi, A., Robinson, S., Sockalingam, S. Evaluation of a Patient-Care Planning Intervention to Improve Appointment Attendance by Adults After Bariatric Surgery. Canadian Journal of Diabetes, pii: S1499-2671(17)31001-8, 2018. PMID: 30121163 Sources of Research Support: None
Background: Recreational athletes and individuals in certain occupations may utilize supplements with the goal of enhancing their physical performance and strength. These individuals may be reluctant to provide their supplement use history due to the stigma associated with performance enhancing drugs. We report a case of supplement use causing elevated dehydroepiandrosterone sulfate (DHEA-S) levels in a young woman presenting with alopecia. Clinical Case: A 29-year-old Caucasian woman presented for evaluation of progressive alopecia spanning 7 years. She was initially managed by dermatology with topical ketoconazole and clobetasol creams followed by intra-lesional triamcinolone injections. Despite treatment, she had minimal improvement. She reported menarche at age 15 with normal regular monthly menstrual cycles and normal breast development. She has no significant medical history. Physical exam was unremarkable other than localized alopecia and athletic build. She denied any medication use. Routine laboratory screening showed a DHEA-S of 624 ug/dL (ref. 35-430) with repeat DHEA-S at 602.2 ug/dL. Complete blood count, metabolic panel, thyroid function test, total/free testosterone, estradiol, 17-hydroxprogesterone, urinary 17-ketosteroids, prolactin, and iron panel were all within normal limits. Anabolic abuse screen was negative. Adrenal imaging was normal. After discussion on elevated DHEA-S results, patient revealed that she was taking a supplement that contained enobosarm (Ostarine). She was instructed to discontinue the supplement and repeat her labs but she was lost to follow up. Discussion: Developed in 1997, enobosarm is a selective androgen receptor modulator (SARMs) that is increasingly used as a performance-enhancing drug. SARMs have tissue specific androgenic receptor effect and patients on SARMs can present with a completely normal hypothalamic-pituitary gonadal axis without biochemical evidence of hyperandrogenism; which was the case in our patient. Conventional anabolic drug abuse screens do not detect SARMs. There have been multiple studies that have evaluated different performance enhancing supplements and nearly 25% of tested products contained compounds that were not correctly labeled. It is therefore uncertain what may have raised this patient’s DHEA-S levels, but the authors theorize the supplement may have contained exogenous DHEA rather than a direct effect of enobosarm. Unfortunately, there is paucity of data or literature on the effect of SARMs on androgen hormone synthesis and DHEA-S levels. A PubMed search for SARMs and DHEA-S levels led to zero returns. It is important for clinicians to recognize and identify the possibility of SARM or supplement use in order to guide diagnostic and management decisions. Further investigation is needed to understand the impact of SARMs on laboratory data and its long term effects.
The androgen receptor (AR) is a hormone activated transcription factor which is important for the growth and progression of prostate cancer (PCa). Metastatic PCa is treated with androgen deprivation therapy (ADT) based regimen but eventually, these tumours develop resistance through multiple mechanisms that reactivate AR. These include expression of constitutively active AR splice variants that lack a ligand binding domain such as AR-V7. To characterize the common and unique activities of AR and AR-V7, we engineered LNCaP and VCaP cell lines that express AR-V7 in response to doxycycline and performed RNA-Seq. Many genes were found to be regulated by both AR and AR-V7, but there were also genes regulated by only one isoform. Moreover, both isoforms altered mRNA splicing of numerous target genes, in some cases without altering overall levels of mRNA. In many cases, the alterations in splicing are AR isoform specific. To understand the differential splicing by AR isoforms, CD44 mini-gene splicing assay and studies of endogenous target genes are being done. The CD44 mini-gene contains an MMTV promoter and is a multi-functional molecule containing two variable exons, v4 and v5 that can be either included or excluded during the splicing process. Both AR and AR-V7 induce transcription from an MMTV promoter. Co-transfection of AR or AR-V7 with the CD44 mini-gene in Hela cells demonstrated that CD44 splicing was altered by AR and its variant AR-V7. R1881 treated samples showed a shift to RNA products resulting from the skipping of both variable exons. Treatment with R1881 showed a decrease in the PSI (Percent Spliced Inclusion) as compared to obtained in the absence of R1881 or AR-V7. In contrast, AR-V7 minimally induced single exon skipping as compared to vehicle. These observations suggest that AR and its variants can have marked effects on RNA splicing to produce radically different proteins with different splicing/inclusion ratio. Analysis of the RNA-Seq data revealed that multiple types of splicing events are affected by the AR isoforms. For instance, whether an isoform favours exclusion or inclusion of a particular exon is event specific. A comparison of splicing in VCaP and LNCaP models showed that only a subset of events were common to the two models. One of the most differentially regulated events in both lines is an exon inclusion/exclusion event in PGAP2. Both isoforms increase the inclusion/exclusion ratio. However, further analysis showed that AR-V7 preferentially increased the levels of inclusion, but did not affect the basal levels of skipping. Also, AR increased the inclusion/skipping ratio through repression of expression of the skipped product. Similar analyses for other splicing events are ongoing. Our studies will provide a better understanding for the molecular basis for isoform specific actions and the role of AR/AR-V7 in the regulation of mRNA transcription and splicing.
Supra-physiological Testosterone Treatment Reduces Aortic Atheroma but Increases Markers of Cardiac Hypertrophy The impact of testosterone on the cardiovascular system is controversial and concerns have been raised in regard to the safety of testosterone replacement therapy (TTh) in hypogonadal men with cardiovascular disease. We have examined the effects of supra-physiological concentrations of testosterone upon aortic fatty streak formation in high-fat diet-fed testicular feminized (Tfm) mice, which exhibit non-functional androgen receptors (AR) and low circulating testosterone levels. Tfm mice were fed a high-cholesterol diet ad libitum for 28 weeks and received fortnightly intramuscular injections of either saline (Tfm, n=8, XY, n=8), 10uL of 250mg/mL testosterone alone (Tfm, n=8, XY, n=8), or in conjunction with either a 30uL intramuscular injection of 50mg/ml estrogen receptor-alpha antagonist fulvestrant (Tfm, n=8), or with 10mg/kg/day of aromatase inhibitor anastrazole in drinking water (Tfm, n=7). Cardiac tissue mRNA was analysed by qPCR for markers of hypertrophy (ANP, BNP, alpha-actin, MYH7, Gsk3-beta, SM22-alpha). A significant reduction in aortic fatty streak formation was observed in Tfm mice receiving supra-physiological testosterone compared to those receiving saline; 1.25+0.36% versus 2.85+0.37% respectively (p<0.01). No significant increase in aortic lipid deposition was observed in XY littermates administered supra-physiological testosterone compared to those receiving saline; 0.22+0.13% versus 0.32+0.12% respectively (p=ns). Aortic fatty streak formation in Tfm mice receiving supra-physiological testosterone in conjunction with fulvestrant or anastrazole was similar to that observed in Tfm mice receiving supra-physiological testosterone therapy alone; 0.71+0.18% and 1.09+0.81% respectively versus 1.25+0.36% (both p=ns) indicating non-genomic actions of testosterone. Hypertrophic markers and heart weights were elevated in XY littermates receiving supra-physiological testosterone but not in Tfm mice compared to placebo suggesting AR-dependent actions. This evidence suggests that supra-physiological levels of testosterone protect against diet induced aortic atheroma formation, at least in part, via non-genomic mechanisms but incur increased cardiovascular risk by potentially inducing cardiac hypertrophy via AR-dependent actions. Dose titration to physiological levels and careful monitoring of patients throughout treatment is key to eliciting the beneficial cardiovascular effects of TTh in hypogonadal men.
Hormonal therapies effectively reduce the frequency and severity of vasomotor symptoms (VMS) in menopausal women; however, whether the effect is clinically meaningful to women is typically not determined. Oral estradiol/progesterone (E2/P4; mg/mg) 1/100 and 0.5/100 significantly improved moderate to severe VMS versus placebo at weeks 4 and 12. The objective of these analyses was to determine the clinical importance (meaningfulness) of E2/P4 treatment versus placebo in menopausal women. REPLENISH, a phase 3, randomized, double-blind, placebo-controlled trial, evaluated the safety and efficacy of E2/P4 oral capsules in symptomatic, postmenopausal women with a uterus. Clinically meaningful reductions in weekly VMS frequency were determined using 3 patient-reported outcomes as anchors (VMS severity score, clinical global impression [CGI], and question 1 from the vasomotor domain of the menopause-specific quality of life questionnaire). The proportion of women who had a clinically important response with 0.5/100 was compared with placebo using the Fisher’s exact test. Spearman correlations were also performed across the 3 anchors. Clinically meaningful reductions in weekly VMS frequency ranged from 32 to 43 at week 4, and from 32 to 48 at week 12. Significantly more responders were observed with 0.5/100 than with placebo for all 3 anchors at both weeks 4 (all, P<0.05) and 12 (all, P≤0.002). All 3 anchors were correlated, supporting their acceptability as appropriate anchors. Treatment with E2/P4 0.5/100 provided consistent clinically meaningful improvements in the weekly frequency of moderate to severe VMS in menopausal women, similar to what has been observed with the CGI-anchor for E2/P4 1/100.
Peripartum and postpartum depression can be detrimental to both the mother and the developing child. Use of antidepressants, such as selective serotonin reuptake inhibitors (SSRIs), is common during the peripartal period and SSRIs have been the leading prescribed antidepressant to treat maternal depression. One of the most commonly prescribed SSRIs is sertraline (Zoloft®) because of the limited fetal teratogenic effects observed, unlike maternal paroxetine (Paxil®) usage which can manifest in fetal cardiovascular defects. Fluoxetine (Prozac®), like sertraline, has previously been shown to have limited teratogenic effects, however, we have shown treatment with fluoxetine for the entire period of pregnancy and lactation in mice compromises pup bones at weaning resulting in decreased long bone length and head circumference. Furthermore, maternal fluoxetine usage results in a sustained reduction in maternal bone mineral density post weaning, which may lead to long-term osteopenia, putting the mother at risk for bone-related disorders later in life. We hypothesized sertraline, like fluoxetine, will compromise maternal bone postpartum and fetal bone development at weaning. Treatment with sertraline in C57BL/6 dams throughout pregnancy and lactation reduced litter size (5.4 pups/dam) and increased pup mortality during the first 24 hours postpartum (20% dead pups/litter) compared to controls (6.8 pups/dam, 5% dead pups/litter, respectively; P < 0.018). Maternal calcium transporters (Orai1 and Serca2) were downregulated in the mammary gland in sertraline-treated dams on day 21 of lactation (P < 0.0032). Together, our data suggests in utero pharmacological exposure to sertraline may induce a failure to thrive in the pups and alters calcium metabolism in the dam. SSRI exposure during pregnancy and lactation may adversely affect the developing neonate(s) as well as have lasting impacts on the mother.
Vaginal bleeding associated with hormone therapy (HT) is the most common reason for treatment discontinuation. Cumulative amenorrhea rates (no bleeding or spotting for 13 cycles) ranging from 23% to 49% with current HT options¹⁻³ are relatively lower than the 56% to 73% observed in the REPLENISH trial.⁴ The REPLENISH trial (NCT01942668) was a 12-month, phase 3, randomized, double-blind, placebo-controlled, multicenter trial that evaluated TX-001HR (bioidentical 17β-estradiol and progesterone [E2/P4] combined in a single, oral softgel capsule; 1 mg E2/100 mg P4 FDA approved as BIJUVATM; TherapeuticsMD, Boca Raton, FL) vs placebo in 1835 menopausal women (40−65 y; intact uterus) for the treatment of menopausal, moderate to severe vasomotor symptoms (VMS). Overall, the proportion of women with no bleeding was high (74%-90%) with up to 1 year of TX-001HR. The objective of this analysis was to determine predictors of vaginal bleeding. Univariate analyses were used to assess the impact of age, race, BMI, smoking, time since last menstrual period (LMP), age at LMP, tubal ligation, parity, baseline E2 concentration, and baseline frequency and severity of VMS (mild=1 to severe=3) on the incidence of vaginal bleeding at any time during the study (cumulative incidence at cycle 13) and for bleeding that occurred in the first 3 months of the study (cumulative incidence at cycle 3). Predictors of vaginal bleeding with HT (at cycle 13) were age, time since LMP, E2 concentration, and severity of VMS. The likelihood of bleeding was reduced by 42% for every 5-year increase in age (P<0.0001) and by 39% for every 5-year increase in the time since LMP (P<0.0001). Incidence of bleeding significantly decreased by 54% with baseline E2 levels <10 pg/mL (P=0.0001), 59% with E2 levels <5 pg/mL (P<0.0001), and 47% for E2 levels 5 to <10 pg/mL (P=0.0040), when compared with E2 levels ≥10 pg/mL. There was 33% less bleeding reported for women with less severe VMS (<2.5 points). No statistically significant differences in reports of bleeding were noted for race, BMI, smoking, age at LMP, tubal ligation, parity, or baseline frequency of moderate to severe VMS. Similar predictors were noted for bleeding at cycle 3, except for baseline VMS severity, which was no longer a significant predictor. Cumulative amenorrhea was relatively high in TX-001HR users. Vaginal bleeding in women treated with TX-001HR increased in women who were younger, experienced their LMP more recently, and had higher baseline E2 concentrations. Even though the rate/amount of bleeding was relatively low overall,⁴ these data may help clinicians inform women who may be more likely to experience vaginal bleeding while taking combined E2/P4 for menopausal symptoms. 1. Prempro tablets PI. Wyeth Pharmaceuticals. 2. Activella tablets PI. Novo Nordisk FemCare AG. 3. Angeliq Tablets PI. Berlex. 4. Lobo RA, et al. Obstet Gynecol. 2018;132:161-170.
INTRODUCTION: Oocyte number and quality are known to decline with age. However, fertility varies significantly even among women of the same age. Given that maternal age has been delayed in recent years, an ovarian biomarker that could reflect follicular activity with precision and accuracy is needed in reproductive medicine. In recent years, two key methods, the concentration of serum antimüllerian hormone (AMH), which reflects the number of small antral follicles and is predictive of ovarian response, and antral follicle count (AFC) performed by ultrasonography, have emerged as preferred methods for assessing ovarian reserve. AIM: To assess the influence of women’s age in the association between AMH serum level and antral follicle count by ultrasonography in the evaluation of ovarian reserve SUBJECTS AND METHODS: 49 women between 25 and 45 years old who attended our laboratory with request for AMH and transvaginal ultrasound in early follicular phase were included in the study. In all of them serum AMH was tested using an electrochemiluminescence immunoassay (ECLIA) on a Roche Diagnostic Cobas e801 analyzer. Transvaginal ultrasonography follicle count was performed in both ovaries by Philips affinity 70 on first days of the menstrual cycle. Statistical analysis was performed through SPSS 23 software. RESULTS: Median and ranges of the variables are the following: AMH: 0.78 (<0.03-9.98) ng/ml and AFC: 6 (1.0-60.0) follicles. AMH and AFC were negatively associated with age (r: -0.302, p< 0.01; r: -0.267 p<0.01, respectively). AMH showed a positive correlation with AFC (r=0.567,p<0.01). We then divided the study population in two subgroups, according to age: Group 1, women <40 years old(n=28) and Group 2, women ≥40 years old (n=21).Considering AMH= 1ng/ml and AFC = 7, the cut-off value used routinely in our institution, we calculated the Kappa coefficient in each group to test the degree of agreement between these two variables, with the following results: Group 1, Kappa= 0.4510, CI 95% [0.1566 – 0.7453], p= 0.0088; Group 2, Kappa= -0.0370, CI 95% [-0.4371 – 0.3630], p=ns. CONCLUSION: despite the positive correlation found between AMH levels and AFC in the whole group, Kappa values show that in women younger than 40 years serum AMH>1 ng/ml is a good predictor of AFC >7, but this agreement is lost in women above this age, with the cut-off values used in this study. These results must be confirmed with a larger group of women.
Context: A strong association between excess visceral fat and the presence of metabolic factors, hypertension and type 2 diabetes have been demonstrated. Patients with primary aldosteronism (PA) are complicated by metabolic syndrome more frequently than those without PA. Aldosterone-induced mineralocorticoid receptor activation has been reported to impair insulin sensitivity in adipocytes and skeletal muscle. An excess of visceral fat has been hypothesized to cause an elevation of aldosterone secretion, and to cause insulin resistance in patients with PA. Objectives: To clarify the role of visceral fat in the pathophysiology of PA, we investigated the correlation of visceral fat parameters with plasma aldosterone concentration (PAC), and the correlation of visceral fat parameters with homeostasis model assessment insulin resistance (HOMA-R) in patients with PA. Materials and methods: This retrospective observational study comprised 131 patients diagnosed with PA between 2007 and 2017 at Sapporo City General Hospital. We divided participants into two subtypes, aldosterone producing adenoma (APA, n = 47) and idiopathic hyperaldosteronism (IHA, n = 84), utilizing adrenal venous sampling. We excluded patients with suspected autonomous cortisol secretion, defined as serum cortisol levels ≧ 3 μg/dL after a 1-mg dexamethasone suppression test. We analyzed the correlations of visceral fat percentage (VF%), visceral fat area (VFA) evaluated by computed tomography studies and PAC, and the correlations of VF% and VFA with HOMA-R in each subtype group. Results: Age and sex distribution was not different between patients with APA and patients with IHA (p = 0.85, p = 0.18, respectively). PAC was significantly higher in patients with APA than patients with IHA (p < 0.001). Serum potassium levels were significantly lower in patients with APA than patients with IHA (p < 0.001). Body mass index was not different between these two subtypes (p = 0.87). VF% was significantly higher in patients with IHA than in those with APA (p = 0.02). The number of patients treated as type 2 diabetes mellitus with oral medicine, HbA1c, and HOMA-R were not different between these two subtypes (p =0.76, p =0.22, p = 0.12, respectively). Patients with IHA showed a positive correlation of VF% with PAC (r = 0.377, p < 0.001), and VFA with PAC (r = 0.443, p < 0.001). In contrast, patients with APA showed no significant correlation of VF% and VFA with PAC. Patients with APA and patients with IHA showed a positive correlation of VF% and VFA with HOMA-R. Conclusions: Between two subtypes of PA, the association of visceral fat on insulin resistance was not different. The association of visceral fat on PAC was different in two subtypes of PA. Only in patients with IHA, visceral fat may play a role in regulation of aldosterone secretion.
The ability of cancer cells to metastasize is the pivotal step that allows a primary tumor to become a life-threatening malignancy. This mechanism is coordinated by transcription factors driving cancer cells to acquire a mesenchymal state through a process called epithelial-mesenchymal transition (EMT). The study of potential therapeutic markers that can prevent or even reverse this phenomenon in metastatic cancers is crucial. To this effort, we have focused on studying the role of DAX-1 (Dosage-sensitive, sex-reversal, adrenal hypoplasia congenita, on the X chromosome, gene 1) as a transcriptional repressor of genes involved in EMT. DAX-1 is a member of the nuclear hormone receptor superfamily with key roles in adrenal and gonadal development. However, recent studies have focused on studying DAX-1 transcriptional regulation of genes involved in cancer progression. We hypothesize that DAX-1 is able to repress the transcription of genes involved in EMT leading to a reduction of metastatic rates in prostate cancer cells. We found that introducing DAX-1 into a prostate cancer cell line that lacks endogenous expression of DAX-1 was sufficient to repress the transcription of EMT markers and lead to a drastic reduction of metastatic rates. Parallel to these findings, we used a CRISPR KO model of DAX-1 to study its role in hormone-dependent prostate cancer. We have previously shown that treatment of hormone-dependent prostate cell lines with a non-aromatizable androgen leads to up-regulation of DAX-1 expression. Here, we use the same mechanism to analyze DAX-1 and AR regulation of metastatic markers in an androgen induced DAX-1 model in parallel to an androgen stimulated DAX-1 KO model. Results have shown that DAX-1 is sufficient to repress the expression of metastatic markers and reduce metastatic rates in prostate cancer cells. These results shed light into the convoluted web of biological processes coordinating prostate cancer progression and could reveal new therapeutics for the treatment of prostate cancer.
*RC and RC contribute equally as senior authors. Introduction:Diabetes management technologies are rapidly evolving and devices such as Continuous Glucose Monitors (CGM’s) and insulin pumps are becoming more sophisticated. These technologies are being covered by insurance companies and, in the near future more patients are expected to use these devices. Therefore, endocrinologists and endocrinology fellows need to feel competent in the indications, use and interpretation of output from these devices. Objectives: To our knowledge, no prior research has explored the prevalence of wearing CGM’s and/or insulin pumps among in-training fellows and their perception of doing so as part of their education. Our objectives therefore are: to estimate the frequency with which wearing a CGM and/or insulin pump is used as a learning opportunity and to explore the main motivators and perception on its value. Methods: Amultiple-choice survey that addressed fellow’s level of training, type of fellowship training program, and use of CGM and/or insulin pump among fellows was sent to all ACGME endocrinology, diabetes and metabolism program coordinators or programs directors. We asked them to forward this survey to their graduating fellows. Their perception on the value of wearing these devices was addressed. Results: 51 graduating fellows responded to the survey. 78.43% and 62.5% of them wore a CGM and insulin pump respectively. 89.48% and 90% of those who wore a CGM and insulin pump respectively thought it was above average value for their education and the most common reasons were to learn the technical aspects and understand what patients with diabetes go through. Conclusions: Wearing a CGM and/or insulin pump is perceived by endocrinology graduating fellows as valuable to their education specifically to learn the technical aspects, understand the patient’s experience and develop empathy.
Background: Polycystic ovary syndrome (PCOS), a metabolic and reproductive associated disease, defined as hyperandrogenism with reproductive dysfunction including menstrual disorder, anovulation, infertility, polycystic ovary and so on. We previously showed reported a high percentage of activating autoantibodies (AAb) directed toward the second extracellular loop (ECL2) of gonadotropin-releasing hormone receptor (GnRHR) in PCOS patients, and further demonstrated elevated GnRHR-autoantibody (GnRHR-AAb) could induced insulin resistance in energy storage and peripheral tissue in immunized animals. In the present study, we have now induced specific GnRHR-ECL2 AAb in rats and explored the underlying mechanisms of their resultant reproductive dysfunction. Methods: Sixteen SD rats were randomly divided into 2 groups: a GnRHR group (n=8) and a control group (n=8). Rats in the GnRHR group were immunized with GnRHR ECL2 peptide while the controls were not. Epitope mapping of GnRHR-ECL2-directed AAb was performed using octapeptide multipin solid-phase peptides. Rat estrus cycle was measured through pudendum appearance and vaginal smears. Ovarian and pituitary tissues were collected to observe ovarian morphological changes, to examine the expressions of proteins and genes of insulin signaling pathway by Quantitative real-time PCR respectively. The concentration of inflammatory cytokines in the ovary was detected by Bio-plex Pro™ magnetic bead-based assays on the Bio-plex®. Results: The GnRHR-AAb titers and activity in the GnRHR group were significantly higher than the control group, and the GnRHR-AAb from the immunized rats reacted predominantly with the peptide sequence FSQCVTHC of the GnRHR-ECL2. Numbers of LH pulses and concentration of testosterone in GnRHR group were significantly higher than control group. The GnRHR group exhibited lower frequency of in the appearance of proestrus and estrous phases while the control group represented had a higher frequency in the appearance of metestrus and diestrus stages on estrus cyclicity. The GnRHR-immunized group showed demonstrated increased atretic follicles, decreased corpora lutea, loosely packed granulosa cells, and thecal cell hyperplasia in ovarian tissue compared with controls group. There was GnRHR group represented increased expressions of IRS-1, PI3K and GLUT-1 in ovarian and pituitary tissues compared with control group. However, no obvious changes of inflammatory cytokines are observed in ovarian tissues between two groups. Conclusion: Chronic elevated GnRHR-AAb exerts induced reproductive dysfunction through increased ovarian LH secretion and androgen production, thus likely leading to compensatory hyperinsulinemia which ultimately enhanced insulin signaling in reproductive tissues to exert more and androgen production to, which may provide a novel etiological mechanism for PCOS.
Background: Sertoli-Leydig cell tumors account for less than 1% of ovarian tumors, and information about their biochemical markers has been lacking. Objective: The objective was to characterize the hormonal profile of Sertoli-Leydig cell tumor, which should be helpful in recognizing this rare condition in the future. Methods: We reviewed test results including serum total and free testosterone, steroid hormone precursors, and inhibin B levels in a 17-year-old adolescent girl with ovarian Sertoli-Leydig cell tumor who developed secondary amenorrhea for 6 months, deepening of the voice, acne, and severe hirsutism. Results: Our patient had serum testosterone 641 ng/dL (expected 20 - 38), dihydrotestosterone 42.5 ng/dL (expected 3 - 18), 17-OH progesterone 659 ng/dL (expected 20 - 265), androstenedione 869 ng/dL (expected 50 - 224), 17-OH pregnenolone 760 ng/dL (expected 53 - 357), DHEA 1250 ng/dL (expected 4 - 491), and DHEA-S of 366 mcg/dL (expected 44 - 248). Inhibin B level was 321 pg/mL (expected <136); inhibin A was normal. Anti-mullerian hormone, a-fetoprotein, carcinoembryonic antigen, and CA-125 tumor markers were not elevated. Karyotype was female 46,XX. Dexamethasone 0.5 mg QID PO for 4 days resulted in plasma ACTH <5.0 pg/mL and serum cortisol <1.0 mcg/dL, total testosterone 611 ng/dL, free testosterone 25.1 ng/dL (expected <0.04 - 1.09 ng/dL), and 17-OH progesterone 887 ng/dL. Abdomen and pelvis MRI demonstrated a right ovarian mass primarily solid with high cellularity, measured 4.4 x 3.9 cm; there was at least moderate diffuse enhancement of the mass after contrast administration; adrenal glands were normal. Surgical pathology of the resected right ovary revealed moderately to poorly differentiated Sertoli-Leydig cell tumor. Single antibody immunostain procedures with appropriate controls showed a staining pattern supportive of this rare diagnosis: WT-1 showed moderate nuclear staining, calretinin showed a strong positive stain, and CK showed a patchy moderate staining pattern; immunostains for myogenin, desmin, and EMA were negative. Genetic testing revealed a germline heterozygous mutation in DICER1 gene, c3737del, p.Asn1246Metfs*12, establishing the diagnosis of DICER1 syndrome, an autosomal dominant disorder predisposing to cancer. Menses resumed one month after tumor resection. Conclusions: High serum 17-OH progesterone, androstenedione, 17-OH pregnenolone, and DHEA levels used as indicators of adrenocortical function could be markers of an ovarian tumor. If serum 17-OH progesterone and testosterone remain high when cortisol and plasma ACTH are suppressed on Dexamethasone test, a source of 17-OH progesterone and testosterone is other than ACTH-dependent adrenal one. High serum inhibin B level may be sign of an ovarian tumor. Patients with Sertoli-Leydig cell tumor should be screened for DICER1 gene syndrome to assess risk for other rare neoplasms.
Introduction: Although potential risk of DKA, ketogenic diet became popular among T1DM patients as proven to reduce the incidence of hypoglycemia, glycemic variability, and HbA1c (1). Severe carbohydrate restriction had not been included among DKA precipitating factors in pregnancy since a minimum consumption of 175 g of carbohydrates a day is recommended for pregnant women by the IOM and ADA. Proband: A 35-year-old pregnant woman (G2P1 at 29 weeks’ gestation) was admitted to the ICU because of a 12-hour history of severe nausea, 4 vomiting episodes (stomach contain), weakness, and metabolic acidosis accompanied by low normal BG values for pregnancy. She had a history of bulimia nervosa since 15 years of age that was well-controlled by severe carbohydrate restriction prior to pregnancy, and T1DM since 19 years of age. Her pre-pregnancy carbohydrate daily intake was ~ 20–30 g and BMI 24.1 kg/m2. Good glycemic control was established before conception and continued until delivery. Upon admission, she was tachypneic and suffering from diffuse abdominal pain. Fetal monitoring and abdominal ultrasonography showed no significant changes, and the sonographic fetal weight was appropriate for gestational age. The laboratory results revealed metabolic acidosis pH 7.23, bicarbonate 11.7 mmol/L, anion gap 22.5, and BG 78 mg/dL. Lactate and salicylate blood levels were normal. The blood β-hydroxybutyrate was highly positive and the patient was diagnosed with euglycemic DKA. She was managed with a continuous intravenous insulin infusion and fluid and electrolytes repletion. The patient was discharged after three days of hospitalization only to return four days later because of the similar complaints with findings of blood β-hydroxybutyrate 7.7 mmol/L and BG levels of 61–65 mg/dL. Because of persistent ketonemia with low normal BG levels, patient was questioning about her dietary habits and revealed to maintain severe carbohydrate-restricted pre-conception eating manners. After repeated consultations by specialists for eating disorder, patient agreed to increase her carbohydrate intake to 120 gr per day. Fetal brain MRI, fetal heart and brain ultrasound were performed to evaluate potential negative effects of ketones, and all were found normal. No major malformations were observed in the newborn, and a normal growth pattern was observed at 5 months of age. Conclusion: This is the first report of a pregnant T1DM patient with long lasting bulimia nervosa excellently controlled by severe carbohydrate restriction prior pregnancy and occultly continued against medical advice through the pregnancy, causing repeatedly hypoglycemic-euglycemic DKA. References: 1. Schmidt S, Christensen MB, Serifovski N, et al. Low versus high carbohydrate diet in type 1 diabetes: A 12-week randomized open-label crossover study. Diabetes Obes Metab 2019; 21:1680- 1688
Prostate cancer (PC) is the most commonly diagnosed cancer among males in the US. The standard of care for patients with metastatic PC is based on androgen-depletion therapy (ADT), including a first-line chemical castration, and a second-line, treatment with high-affinity antagonists for androgen receptor (AR), e.g. enzalutamide (Enz). Unfortunately, most of the patients eventually relapse with castration-resistant metastatic PC (CRPC). Further, use of Enz is thought to cause a growing concern for prevalence of highly aggressive AR-independent CRPC. Therefore, the molecular mechanisms of Enz-resistant (EnzR) CRPC are need to be understood urgently in order to identify novel targeted therapies for deadly CRPC. To approach Enz-resistant CRPC we have created EnzRPC cell lines by passaging cells in the presence of sub-lethal doses of Enz for over 6 months Analyzing the global profiles of expressed genes, we determined chemokine receptor CXCR7 (also known as atypical chemokine receptor ACKR3) as one of the top upregulated genes in EnzRcells. Utilizing molecular techniques we found that AR directly binds to promotor and enhancer regions of CXCR7 gene and inhibits its expression. EnzR PC cells depends on CXCR7-activated signaling in order to survive anti-AR therapy in vitroand in vivowhen injected in immuno-deficient mice. Moreover, we found that patients with metastatic CRPC increase CXCR7 expression comparing to localized PC tumors, and patients who relapse on second-line ADT further increase CXCR7 expression. CXCR7 is a 7-transmembrane (7TM) receptor, which when activated recruits ARRB2, a cytoplasmic scaffold protein. This leads to receptor internalization and activation of MAPK kinases. Normally, CXCR7 is activated by CXCL12 (SDF1a), CXCL11 (ITAC), and MIF. However, we observed that in EnzR PC cells upregulated CXCR7 signaling is constitutive, causing sustained ERK activation. Thus, when we combined MAPKK small molecule inhibitor with enzalutamide we observed superior therapeutic efficacy for CRPC in pre-clinical studies. Besides MAPK, CXCR7 can cross-activate other pro-survival signaling cascades, such as Akt and EGFR. Moreover, GSEA analysis reveals that EnzRPC cells further reduce AR signaling upon CXCR7 knock down suggesting another level of communication between CXCR7 and AR. Currently, CXCR7 lacks known potent antagonists, which would entirely cancel downstream signal activation, thereby, we are searching for novel small molecule CXCR7 inhibitors. Taken together our data suggest that AR and CXCR7 form a multilevel signaling axis, important for EnzR CRPC progression. Currently, we are focusing on further understanding the molecular mechanisms of CXCR7 tumor-promoting activity. In parallel we are developing novel anti-CXCR7 therapies that will be translatable into the clinics as a targeted therapy for patients with deadly CRPC.
Glucocorticoids (GCs) are used in the treatment of chronic inflammatory diseases due to their potent anti-inflammatory actions. Their use is limited due to major systemic side effects including osteoporosis and muscle wasting. This is further complicated by pre-existing inflammatory muscle wasting and bone loss, driven by pro-inflammatory pathways that are themselves directly suppressed by GCs. Understanding the interaction between these processes and their net effects on bone and muscle metabolism in vivo is critical in informing our approach to managing these patients. We investigated this in the TNF-tg model of chronic inflammation receiving the GC corticosterone (100 μg/ml) at therapeutic doses in drinking water over 3 weeks. Arthritis severity and clinical parameters were scored, front paws and tibias assessed by μCT analysis and markers of bone metabolism and inflammation determined by serum ELISA. Muscles were weighed and taken for histological analysis. Metabolic and inflammatory gene expression were determined by RT qPCR. Corticosterone potently suppressed clinical scores of inflammation and circulating pro-inflammatory cytokines including IL-6 in TNF-tg mice. A decrease in pro-inflammatory gene expression was seen in bone and muscle. Analysis of trabecular bone volume (BV/TV) and trabecular number (Tb.N) by μCT revealed that TNF-tg mice receiving corticosterone were protected from bone loss relative to controls (BV/TV: TNF-tg 2.19% ± 0.2 vs TNF-tg CORT 4.25% ± 0.2, P≤0.001; Tb.N: TNF-tg 0.0004 1/μm ± 0.00004 vs TNF-tg CORT 0.0008 1/μm ± 0.00003, P≤0.001). Whilst serum markers of bone formation (P1NP) and osteoblast gene expression were significantly suppressed in mice receiving corticosterone, an overwhelming suppression of osteoclast activity determined by serum CTX-1 and osteoclast numbers appeared to mediate the protective actions of GCs in the TNF-tg mouse (CTX-1: TNF-tg 81.6 ng/ml ± 10.7 vs TNF-tg CORT 36 ng/ml ± 2.7, P≤0.01). In contrast, in TNF-tg mice receiving corticosterone muscle wasting was further exacerbated. Muscle weights were significantly reduced in TNF-tg mice receiving corticosterone, with muscle fibre size markedly suppressed (Quad: TNF-tg 0.15 g ± 0.012 vs TNF-tg CORT 0.10 g ± 0.009, P≤0.01; TA: TNF-tg 0.06 g ± 0.005 vs TNF-tg CORT 0.03 g ± 0.004, P≤0.01). This was driven by a marked upregulation of anti-anabolic and catabolic gene expression in TNF-tg mice receiving corticosterone relative to vehicle treated controls (FBXO32; 4.7 fold, p<0.001, TRIM63; 3 fold, p<0.05, REDD1; 4 fold, NS, FoxO1; 3 fold, p<0.01). In the TNF-tg model of chronic inflammation, therapeutic GCs prevented local and systemic bone loss but markedly exacerbated muscle wasting. These results suggest that interventions that preserve muscle mass and function should be prioritised where therapeutic GCs are utilized to treat chronic inflammation.
INTRODUCTION: The inadequacy of formative feedback (FF) in graduate medical education (GME) has been a concern of physician-educators and trainees for more than three decades. FF in GME is most often obtained through two assessments, the global ratings scale and In-Training Exam (ITE), both with well documented limitations. The global ratings scale (i.e. “milestones”) is subject to numerous threats to validity including limited direct observations of trainees, rater bias, inaccurate recall, and lack of specific clinical skills to rate. The ITE suffers from construct underrepresentation as atypical clinical scenarios and basic science knowledge is frequently assessed. The multiple-choice format can result in cueing, seem artificial, and removed from real situations. The structured oral exam (SOE), used for FF, does not have limitations of the assessments noted above. To the best of the authors’ knowledge, there have been no published reports of the use of a SOE to provide FF in GME. OBJECTIVE: To determine the educational impact, as well as the feasibility, cost, and acceptability of a SOE for FF of Pediatric Endocrinology (PE) fellows. METHODS: Four cases (precocious and delayed puberty, PCOS, congenital hypothyroidism) were developed for this pilot SOE. Each question and scoring rubric was evidence-based and peer-reviewed by four PE attending physicians. A 5-point Likert scale survey with short answers was developed utilizing best practice guidelines to address our research question. Three faculty/fellow dyads completed the pilot. RESULTS: With regards to educational impact, all fellows and faculty reported that the exam was helpful for both identifying previously unrecognized knowledge deficits, and for providing guidance for the fellow’s future learning, with the majority stating it was quite helpful or extremely helpful. There were no significant concerns regarding need for specialized training to administer the exam, clarity of questions, subjectivity in scoring, or stress on the fellow. Several participants proposed shorter, more frequent sessions to enable longitudinal assessment. An estimated 4 hours of faculty time would be required per fellow per year to administer our comprehensive SOE, consisting of 35 cases, over the course of a 3 year fellowship. Administration by senior fellows was suggested if faculty time is limited. CONCLUSIONS: To the authors’ knowledge, this is the first description of a SOE as a FF tool in GME. Fellows and faculty found this SOE to be educationally impactful, however, modifications could further improve it’s feasibility, cost, and acceptability. The full-scale, multi-center study will provide further investigation of this SOE as a FF tool for PE fellows.
Objectives: The onset of parturition in pregnant women with obesity is frequently delayed. Without induction, these women are nearly twice as likely as normal-weight to have prolonged pregnancy (≥41 weeks gestation) which is concerning because of associated two-fold increased risk of third-trimester stillbirth. Data from vascular studies have shown that different adipokines have different effects on smooth muscle contractility; either as relaxants or constrictors. However, only few studies have investigated their role in uterine contractility, a relationship that we sought to investigate. Materials and Methods: Total of 22 pregnant women scheduled for term cesarean delivery (CD) were recruited. Strips from the first two participants were used to identify dose response effects for each adipokine, and 20 participants’ data were included in the final analysis. Study groups consisted of normal-weight (N=10) and women with obesity (N=10). Myometrial strips were obtained from the hysterotomy incision at the time of the CD. Muscle strips were mounted within experimental recording baths. Both spontaneous and oxytocin induced contractions were recorded by a custom-build data acquisition software. Adipokines of interest included adiponectin, TNFα, resistin, and omentin. Adipokines were added to the muscle baths after muscle equilibration was achieved. Contractions outcomes of interest included forces, durations, and frequencies. Data comparisons were conducted using Wilcoxon Rank-Sum tests; medians and ranges are presented. Results: Forces of contractions in normal-weight participants were double those studied from participants with obesity (13.9 [9.3-34.3] vs. 8.9 [4.8-23.6], p=0.05). There were no statistically significant differences between contractility outcomes of interest after adding adiponectin, TNFα, and resistin to the muscle baths within and between the study groups. In participants with obesity, compared to baseline, omentin significantly reduced the force of spontaneous induced contractions (p=0.002) and prolonged the period between contractions (p=0.01). Importantly, that effect was not seen in normal-weight participants or in oxytocin induced contractions. Omentin also significantly reduced the forces of spontaneous induced contractions (2.9 [2.2-4.6] vs. 14.4 [4.8-33.6]; p=0.01) and prolonged the period (790.6 [753.0-832.0] vs. 611.4 [128.3-702.7]; p=0.04) in participants with obesity compared to normal-weight participants. Differences were no longer observed after adding oxytocin. Conclusion: In vitro, uterine contractions were reduced in muscle samples prepared from pregnant women with obesity compared to normal-weight counterparts. Omentin may have a role in reduced uterine contractility in pregnant women with obesity and that effect may be corrected by oxytocin administration.
Uterine leiomyomas, commonly referred to as fibroids, are benign, estrogen sensitive smooth muscle tumors that occur in the myometrium of the uterine wall. Leiomyomas are common, as it is estimated that 60% of reproductive-aged women are affected, and 80% of women develop leiomyomas during their lifetime. In fact, uterine leiomyomas are the leading cause of abnormal menstrual bleeding or menstrual pain, as well as the number one reason for hysterectomy. Novel treatment options are necessary as current treatments are limited to anti-estrogen therapy or hysterectomy. Estrogen is known to have an effect on the etiology of leiomyomas, but little is known about the proliferative roles of other hormones in leiomyomas. One hormone of interest is prolactin (PRL) which is primarily secreted from the pituitary to regulate lactation, but has been linked to proliferation in breast cancer, perhaps via local prolactin production in breast tissue. With this background, we examined local PRL production and its effects on leiomyomas. RNA isolation and quantitative PCR of human leiomyoma samples (n=20) relative to adjacent myometrium in the same patients confirmed significant expression of both PRL (p= 0.0028) and dopamine receptor D2, a known regulator of PRL production in the pituitary (p<0.0001), with no difference in prolactin receptor expression. Using both immunohistochemistry and immunofluorescence of human leiomyomas samples, we find increased prolactin expression in leiomyomas when compared to adjacent myometrium or control uteri. These results suggest that leiomyomas contain cells producing PRL, which in turn may promote signaling in smooth muscle leiomyoma cells to regulate proliferation. Accordingly, we find that PRL robustly activates STAT5 and MAPK signaling in the rat leiomyoma cell line ELT3. Functional assays were also conducted to evaluate the ability of PRL to induce migration, invasion and proliferation of ELT3 cells. Together, our findings suggest that local prolactin production in leiomyomas may promote their growth, migration, invasion and proliferation. It is possible that this local production is mediated by the dopamine receptor D2. Thus, anti-PRL therapy or dopamine receptor D2 modulation may prove useful in treating this prevalent and often debilitating disease.
Background Approximately 88% of endocrinologists agreed that intravenous levothyroxine (IV T4) is not medically necessary in euthyroid patients if they are nothing by mouth (NPO) for less than 3 days. A prior 2013 retrospective database analysis at our institution revealed that a majority of IV T4 was prescribed inappropriately for NPO status. Therefore, we sought to develop an intervention to reduce inappropriate IV T4 use among hospitalized adults. Methods A training session was provided to clinical pharmacists and medical providers in areas with high IV T4 utilization rates (SICU, MICU, CICU, medical and surgical floors). Compelling reasons to use IV T4 were highlighted, including myxedema coma, cardiogenic shock, suspected malabsorption, or NPO status for >3 days. A retrospective 1-year database analysis was conducted after the intervention to assess IV T4 utilization rates and new onset of arrhythmias and troponin elevations among hospitalized patients. Rates of inappropriate use and adverse effects associated with T4 therapy were compared prior to and after the intervention with the chi-square test. Results Prior to the intervention, 76 patients (mean age 65 years) received IV T4 during the 6-month study period. Of these, 80% of IV T4 was prescribed inappropriately for NPO status at the cost of $38,423. Approximately 11% and 8% of these patients developed new onset arrhythmias and troponin elevations, respectively. After the intervention, 183 patients (mean age 61 years) received IV T4 at our institution during the 1-year study period. Of these, 56% of IV T4 was prescribed inappropriately for NPO status with no other compelling indication at the cost of $75,936. Approximately 20% and 12% of these patients developed new onset arrhythmias and troponin elevations, respectively. Approximately 31% of patients who had unwarranted IV T4 after the intervention had a history of atrial fibrillation, congestive heart failure, or coronary artery disease. The pharmacist led training intervention significantly reduced inappropriate use of IV T4 (p<0.05), however total cost and rates of new onset arrhythmia and troponin elevations did not significantly change (p>0.05). Conclusion A pharmacist led training intervention designed to educate providers and pharmacists about appropriate indications to use IV T4 among hospitalized adults led to a significant reduction in the use of inappropriate IV T4. Cost did not significantly change despite the reduction of inappropriate IV T4 use, likely due to rising drug prices. Despite a modest decrease in inappropriate IV T4 use, our institution aims to further reduce adverse effects, inappropriate use, and cost through a variety of quality control measures, such as implementing EMR order sets and/or restricting use of IV T4 subject to endocrinology approval.
High grade serous ovarian carcinoma (HGSC), the most prevalent and aggressive form of ovarian cancer, contains abundant receptors for the ovarian steroid hormones, estrogen (ER; 76%) and progesterone (PR; 35%). These receptors are known to contribute to breast and reproductive cancer development. Our understanding, however, of the mechanisms of PR isoform action in the initiation and progression of ovarian cancer is limited. HGSC is thought to originate from the fallopian tube epithelium (FTE), and accumulating evidence suggests that serous tubal intraepithelial carcinomas (STICs) are precursor lesions to most HGSCs. Our IHC analysis of normal FTE and STICs showed expression of both total PR and activated phopho-Ser294 PR (p-PR). Interestingly, STIC lesions had greater intensity of focal nuclear p-PR, indicative of highly active transcriptional complexes. To investigate PR isoform signaling in early stage HGSC, we utilized p53-dominant negative mutant FTE cells to generate stable cell lines expressing either the PR-A or PR-B isoform. Progestin (progesterone; R5020) treatment of 2D adherent cultures revealed functional PR signaling through MAPK-dependent p-PR as well as isoform specific expression of PR target genes encoding adhesion molecules (e.g. HEF1), cell cycle regulators (e.g. FOXO1), and glucocorticoid signaling proteins (e.g. CRISPLD2, NDRG1). Progestin treatment also dramatically inhibited proliferation which was recovered following growth factor stimulus, suggesting that PR signaling may promote cell quiescence (G0). To mimic dissemination of FTE cells from STICs, a 3D spheroid model was established. Interestingly, we observed that PR expression and activation greatly increased the formation, size and number of total spheroids compared to PR negative controls. Cells within these spheroids are predominantly Ki67-negative indicating that they are likely non-proliferative and potentially G0-arrested cells. When the spheroids were embedded into collagen, to mimic tumor cell invasion into the peritoneum, only PR+ spheroids exhibited invasive behavior. Additionally, PR-B+ spheroids were more invasive than PR-A+ spheroids; progestin treatment during 3D formation dramatically increased spheroid invasion. Notably, this is the opposite of our results in 2D cultures, where PR-A+ FTE cells display greater proliferative and migratory phenotypes relative to PR-B+ FTE cells. Taken together, our data suggest that activation of PR signaling promotes and enhances the formation and survival of non-adherent 3D FTE structures. Such effects of progesterone are predicted to influence the shedding, aggregation and dissemination of early STIC lesions that form ovarian and peritoneal metastases. Our findings demonstrate the importance of understanding the impact of steroid hormone receptors, including PR isoforms, on early ovarian cancer development.
Background: Ovarian tumors are divided into non functioning and functioning, within the last group, we can find those with endocrine activity that produce androgenization. Ovarian cell tumors, not otherwise specified (SCT-NOS) is a rare type of ovarian sex cordomal tumor and represents the 60% of this tumors, which compromise less than 0.1% of the ovarian tumors. (1) Clinical Case: We here present a 28 year old woman who was referred to the Endocrine Clinic due to secondary amenorrhea and virilization signs. At the age of twelve a diagnosis of polycystic ovarian syndrome (POS) was made and treated with combined oral anticonceptive (COA). Menses became regular only with medication. Six months after she stopped medication, amenorrhea and virilization signs worsened. Biochemically she had levels of serum total testosterone 6.8 ng/mL (0.02-0.45) and free testosterone 42 pg/mL (0.1-6.4) since only pelvic ultrasonography has been made by physician, a transvaginal ultrasound and abdomen – pelvic CT scan showed a anexial tumor. After analysis of biochemical and imaging results a multidisciplinary team performed a surgical extirpation of the primary lesion, which was diagnosed by histopathology as a tumor of lipoidic cells NOS. A month after the surgery, menses became regular. Conclusion: The purpose of this article is to present the available information about this kind of tumors and the treatment recommended. It is mandatory to follow a correct approach among a multidisciplinary team, in order to get the correct diagnosis at the proper time. (1) Zang L, Ye M, Yang G, Li J, Liu M, Du J et al. Accessory ovarian steroid cell tumor producing testosterone and cortisol. Medicine. 2017;96(37):e7998.
Background Recent evidence suggests that 11-oxygenated C19 steroids may have an important role in the assessment of hyperandrogenism [1]. Two in particular, 11β-hydroxyandrostenedione (11OHA4) and 11-ketotestosterone (11KT) have been implicated in polycystic ovarian syndrome [2] and congenital adrenal hyperplasia [3]. At present there are limited methods available to quantitate these potentially useful analytes and no data is available on their stability. Objectives Here, we sought to develop an LC-MS/MS assay to quantify total serum testosterone (T), androstenedione (A4), 17-hydroxyprogesterone (17OHP), 11OHA4 and 11KT. In addition, we aimed to apply the method to evaluate the stability of 11OHA4 and 11KT. Method An LC-MS/MS assay that measured total serum T, A4, 17OHP, 11OHA4 and 11KT was developed and validated in accordance with FDA guidelines. The method was applied to assess the stability of each analyte in serum over a 3 day period. Seven BD Vacutainer® SST™ tubes were collected from 14 volunteers (6 males, 8 females) into. The tubes were stored unseparated at 20°C (68°F) and retrieved at the following times points: 0, 2, 8, 12, 24, 48 and 72h. Once retrieved, samples were centrifuged, aliquoted and stored at -20°C (-4°F) prior to analysis. Results The performance characteristics of the method were acceptable when assessed against an industry standard. Although 11OHA4 concentrations did not significantly change over the 3 day period, we observed a progressive and statistically significant increase in 11KT concentrations between day 0 to day 3 (p<0.001). This increase was most pronounced between time 0 and 8h with concentrations rising on average 26% (95% CI 18 – 33). Summary We have developed a robust LC-MS/MS assay to quantify T, A4, 17OHP, 11OHA4 and 11KT. Application of the method to assess stability has shown that 11KT concentrations increase in-vitro. This change is likely to be clinically significant and underpins the necessity to standardise sample collection before conclusions can be made about the clinical utility of 11KT. References [1] Pretorius E, Arlt W, Storbeck KH. A new dawn for androgens: Novel lessons from 11-oxygenated C19 steroids. Mol Cell Endocrinol. 2017;441:76-85. [2] O’Reilly MW, Kempegowda P, Jenkinson C, Taylor AE, Quanson JL, Storbeck KH, Arlt W. 11-Oxygenated C19 steroids are the predominant androgens in polycystic ovary syndrome. J Clin Endocrinol Metab. 2017;102:2016-3285. [3] Turcu AF, Nanba AT, Chomic R, Upadhyay SK, Giordano TJ, Shields JJ, Merke DP, Rainey WE, Auchus RJ. Adrenal-derived 11-oxygenated 19-carbon steroids are the dominant androgens in classic 21-hydroxylase deficiency. Eur J Endocrinol. 2016;174:601-609.
Background:Polycystic ovary syndrome (PCOS) is one of the common endocrine disorders affecting young women and has been associated with an increased risk of impaired glucose tolerance and type 2 diabetes mellitus. However, there are very few studies on glucose abnormalities in Indian adolescent girls with PCOS. This study was conducted to determine the prevalence of abnormal glucose metabolism in this patient population. Method: Study group comprised of 106 young females aged 13–18 years. None of them were on metformin at the time of initial visit. Clinical examination along with Anthropometric evaluation was done and along with routine hormonal assessment they underwent a standard 75-g oral glucose tolerance test (OGTT). American Diabetes Association (ADA) criteria were used for diagnosis of diabetes (2 hr value>200mg/dl), Impaired glucose tolerance (2 hour value > 140 -199 mg/dl) and Impaired fasting glucose (blood glucose level of 100–125 mg/dl) Results:Out of 106 girls with PCOS diagnosis;1 girl met criteria for diagnosis of type 2 diabetes mellitus (0.9%).15 had impaired glucose tolerance (14.1%). In addition, 2 girls with impaired glucose tolerance were also noted to have impaired fasting glucose (1.9%).Abnormalities of glucose metabolism had significant correlation with BMI(p-0.02),Waist circumference (p-0.01) and testosterone levels (p-0.02). Conclusions:In our series of adolescent PCOS subjects, we report the prevalence of abnormal glucose metabolism to be 14.1% which is quite significant. Based on our results, we recommend that all adolescent girls with a diagnosis of PCOS should undergo formal oral glucose tolerance testing. Further studies are necessary in this field, so as to make guidelines regarding the timing and frequency of this testing, as well as its utility in the clinical management of these girls.
Introduction: HAIR-AN syndrome is a sub-type of polycystic ovarian syndrome which consists of virilization, insulin resistance and acanthosis nigricans. It may be associated with menstrual irregularity, and hyperandrogenic features such as hirsutism and masculinization. Aim: We report an adolescent girl with HAIR-AN syndrome, T2DM, a very high testosterone level (male range) and primary amenorrhea Case report: A 17-year-old female who had been investigated by pediatric endocrinology since the age of 13 because of hyperpigmentation which was proven to be acanthosis nigricans on skin biopsy. The patient was found to have insulin resistance with initially normal glucose level. She has hirsutism and hypertrichosis. There is no similar condition in her family, she has three siblings all are well. She developed T2DM with at the age of 14. She was started on Metformin 2 gram daily and them pioglitazone 30 mg was added when she was 16 years. She never had menarche. The clinical examination revealed an adolescent girl with normal BP 106/68 mmHg, and BMI 19.6kg/m2. She scored 24 onFerriman-Gallwey hirsutism scoring system. She had severe acanthosis nigricans on both axillae. She also had back and upper limbs hyperpigmentation.Lab tests revealed normal thyroid function tests, prolactin, cortisol, DHEA-S, and 17 hydroxy progesterone. Fasting glucose 7.2, insulin 123 μU/ml (2.6-24.9), c-peptide 964, HbA1c 8.2%. Total testosterone 24.61 nmol/L (0.069-2.715), SHBG 184.9 nmol/L, and Free testosterone index 13.31 (0.51-6.53). Her LH 8.9 and FSH 4.7.Radiological investigations revealed polycystic ovaries on pelvic ultrasound. MRI abdomen showed normal adrenals, and mildly enlarged ovaries with peripherally located follicles consistent with polycystic ovarian syndrome. The patient was started in Diane-35 (cyproterone acetate and ethinyl estradiol) oral pills. She started to have menarche three months after using Diane-35. Her Total testosterone had dropped from 24.61 to 1.69 nmol/L (0.069-2.715), SHBG 579 nmol/L, and Free testosterone index 0.29 (0.51-6.53). She reported that the hirsutism is getting less than before starting the treatment.Conclusion: Primary amenorrhea might be a manifestation of in HAIR-AN syndrome due to sever hyperandrogenism. The management of such condition is challenging. In addition to controlling the metabolic parameters, combined oral pills with antiandrogen effect might be effective.
Glucocorticoids are primary stress hormones that regulate brain function. Aberrant levels of these steroids have been implicated in the pathogenesis of cognitive impairments and psychiatric disorders. Glucocorticoids exert their effects on cells via binding the glucocorticoid receptor (GR) and the closely related mineralocorticoid receptor (MR). A distinctive feature of the hippocampus is that it expresses high levels of both GR and MR. However, the specific roles played by these receptors in mediating the direct actions of glucocorticoids in the hippocampus are poorly understood. To elucidate the in vivo function of hippocampal GR and MR, we employed emx1-cre mice to ablate GR (hippocampal GRKO), MR (hippocampal MRKO), or both GR and MR (hippocampal GRMRdKO) in the hippocampus. The single and double knockout mice were born at the expected Mendelian ratio and survived normally through 12 months of age. Small reductions in body weight were observed for the hippocampal MRKO and hippocampal GRMRdKO mice, but not the hippocampal GRKO mice. RNA sequencing performed on hippocampal RNA revealed major differences in both the basal and glucocorticoid regulated transcriptome in the single and double knockout mice. In behavioral assays, all three mutant mice exhibited reduced anxiety in the marble burying test. However, only the hippocampal MRKO and hippocampal GRMRdKO mice showed decreased anxiety in the elevated plus maze and open field tests. A forced swim test revealed no genotype differences in depression-like behavior. In a conditioned fear test, the hippocampal GRKO mice showed a reduction in context-dependent learning whereas the hippocampal MRKO and hippocampal GRMRdKO mice showed an increase in cue-dependent learning. These findings demonstrate that GR and MR regulate both common and unique signaling pathways in the hippocampus that influence anxiety and learning and memory.
Severe hyperandrogenism in post-menopausal women is rare. It may be caused by either benign or malignant neoplasms of the adrenal or ovary. We report a rare case of a post-menopausal woman with hirsutism associated with virilization due to Leydig cell tumors (LCT) of both ovaries. Case report 61 yo female presented for evaluation of hirsutism. She had also been experiencing increased facial hair growth, deepening of voice, clitoromegaly, alopecia, and acne. Physical examination: normal vital signs. Patient had signs of virilization, including coarse hair along her upper lip, chin, lower abdomen and inner thigh with Ferriman-Gallwey score of 8, acne, and clitoromegaly. She had no signs of acanthosis nigricans or Cushing syndrome. Base line labs: Hemoglobin 16.2 gm/dL (ref 12.0 to 15.5), total testosterone 803 ng/dL (ref 3–41), free testosterone 20.2 pg/mL (ref 0.0–4.2), estradiol 77 pg/mL (<6.0−54.7), estrone 148 pg/mL (ref 7–40), FSH 11.5 mIU/mL (ref 25.8 - 134.8), LH 6.90 mIU/mL (ref 7.7 - 58.5), androstenedione 28 ng/dL (ref 17–99), DHEAS 99.9 mcg/dL (ref 19–205), dehydroepiandrosterone 512 ng/dL (ref 31–701), inhibin A 2.3 pg/mL (ref <5), inhibin B <7.0 pg/mL (ref 00-16.9), 17-alpha hydroxyprogesterone 187 ng/dl (ref <51). Her other serum markers such as anti -Mullerian hormone, alpha-fetoprotein, and hCG were normal. A CT scan of adrenals: normal. Similarly a transvaginal US did not show any ovarian pathology, however MRI of the pelvis showed homogeneous ovarian enhancement bilaterally and based on this information a diagnosis of ovarian hyperthecosis was considered and patient underwent laparoscopic bilateral oophorectomy. Pathology confirmed: LCT in both ovaries. Laboratory values performed 3 months later showed the following values: hemoglobin 14.2 gm/dL, total testosterone 13 ng/dL, free testosterone 1.4 pg/mL, LH 124 mIU/mL, FSH 99mIU/mL, estradiol <5.0 pg/mL. In 6 months she had significant improvement in hirsutism and virilization. Discussion Hyperandrogenism, especially serum testosterone in the male range with rapidly progressive hirsutism or virilization signs in a female indicates tumor etiology. Androgen-secreting ovarian tumors are usually small and often embedded in the ovary. Transvaginal US is useful in the diagnosis of ovarian tumors. However, in our case, transvaginal US failed while MRI scan showed bilaterally enlarged ovaries and with this information patient underwent bilateral oophorectomy. Although 5 cases of bilateral LCT are reported in the literature, LCT is unilateral 95% of the time, the pathogenesis of Leydig cell proliferation and LCT is unclear. In conclusion, androgen secreting tumors should be considered in women (especially in post-menopausal state) with hyperandrogenism and hirsutism. In fact, diffuse stromal Leydig cell hyperplasia and small Leydig cell tumors may be missed on imaging and in some cases only pathology can confirm the result.
The association of estrogens with breast cancer has led to the development of drugs that block the action of estrogens to treat and prevent estrogen receptor (ER) positive tumors. However, many tumors eventually become resistant to these drugs. Future drug discovery requires the identification of novel targets in the ER signaling pathway. The identification of proteins that directly bind to ER may offer new therapeutic targets for the treatment of breast cancer. The orphan receptor, DAX-1 (Dosage Sensitive Sex Reversal Adrenal Hypoplasia Congenita on the X Chromosome, gene 1) has been shown to act as a corepressor of ERα actions in MCF7 breast cancer cells. In this study we investigated the expression of DAX-1 during progressive stages of invasive ductal carcinoma with known ER status. To define the effect of DAX-1 expression in ER positive breast cancer cells, we performed gene expression analysis using PCR arrays that were specific for human breast cancer gene targets, human estrogen receptor signaling targets and tumor metastasis targets. We found that adenovirus-mediated expression of DAX-1 inhibits MCF7 cell proliferation and blocks estradiol activation of specific ER target genes, including cyclin D1. Repression of the cyclin D1 gene in MCF7 cells is associated with the recruitment of DAX-1 to the cyclin D1 promoter. Furthermore, introduction of DAX-1 into MCF7 cells leads to cell cycle arrest and an increase in apoptosis. These results demonstrate that DAX-1 functions as a corepressor of ER action in MCF7 breast cancer cells by preventing the activation of growth promoting genes and decreasing the stimulation of cell proliferation in response to estrogens. These finding could have significant implications for future drug development strategies specifically aimed at ER interacting proteins such as DAX-1.
Telemedicine/Virtual Health modalities of health care delivery are rapidly growing across the healthcare continuum. Telemedicine is a useful extension of a typical outpatient Endocrine practice to improve access to care, reduce travel time for patients and improve patient experience. The author will present a program review of a TeleEndocrine practice in the military using a variety of virtual health modalities: Synchronous video between military clinics and to patients homes, remote home monitoring in diabetic patients and asynchronous care delivery. Virtual consultative and management services are provided from our Military Medical Center to multiple military health systems sites located in Puerto Rico, Kentucky, Georgia and New York where there are no endocrinologists at those military sites. Data from over 300 encounters will be presented in terms of case mix, patient demographics, workload, coding and patient satisfaction. Patient experience was evaluated using a survey instrument to assess various domains of the TeleEndocrine consultation (equipment ease of use, video/audio quality, ability to communicate their clinical concerns, previous use of telehealth, perceptions of care deliveryand others. In our population, over 85% had never done a telemedicine consult in the past and of these respondents, 95% reported being satisfied overall with the TeleEndocrine consultation and 3.5 % reported that they would have preferred seeing an endocrinologist in person. In conclusion, telehealth technology adoption and integration into a general Endocrine practice clinical workflow was achieved using various capabilities and the patient experience with this technology was overall positive.
Background: Many women in Bayelsa State, South-South Nigeria have been presenting with different menstrual disturbances and some with infertility problems that are associated with abnormal uterine bleeding, amenorrhea, dysmenorrhea, premature menopause (primary ovarian insfficiency) and premature syndrome (PMS). It is recognized universally that menstrual disturbances may accompany and most times, succeed endocrine disorders. Objective: The aim of the study was to investigate the various endocrine disorders associated with women of reproductive age experiencing different menstrual disturbances attending the different specialist hospitals in Bayelsa State, Nigeria. This is with the interest of establishing the different hormone disorders and its prevalence in these women of Bayelsa State. Method: A total of 1852 subjects (women with menstrual disturbances) were randomly selected using a questionnaire design containing the information of age, last menstrual date, degree of irregularity, whether on medication for any infertility problem or preventive measures and have had any form of surgery. Excluded from the study were pregnant women, women above 40 years of age, those on infertility and contraceptive medication. Analyses of the different hormones of the hypothalamic - pituitary- gonadal reproductive axis were measured using Enzyme Linked Immunosorbent Assay (ELISA). Results: From the study, it was observed that the menstrual disturbances were accompanied with female reproductive hormone fluctuations; with a high significant (P<0.005) percentage, 79.93% of the women having one form of the endocrine disorders. The study has shown that 63.17% of the studied subjects had hypoestrogenism, 38.66% had hypergonadotropic hypogonadism and a non significant (p>0.05) 1.35% with hypogonadotropic hypogonadism. Analysis of the results also showed 15.12% of the subjects had hypothalamic amenorrhea, 32.02% with hyperprolactinaemia and non significant value of 1.30% with hypoprolactinaemia. From the study, 10.92% of the women with menstrual disturbances studied in Bayelsa State had hyperthyroidism, 4.71% with hypothyroidism, 3.74% with T3 thyrotoxicosis and 2.37% with TBG excess. Conclusion: This study therefore concluded that a significant number (p<0.05) of women in Bayelsa State, Nigeria with menstrual disturbances are associated with endocrine disorders probably due to life style (diet, obesity), environmental factors and an underlying illness(infections, cancer and polycystic ovarian syndrome).
The mechanisms that lead to the initiation of parturition are incompletely defined. Parturition timing is mediated by signals from both mother and fetus. Our previous findings using mice that were double-deficient in steroid receptor coactivators (Src)1 and 2 (Src-1/-2d/d) suggest that the fetus signals its mother when it is ready to be born through fetal lung production of surfactant components, surfactant protein A (SP-A) and platelet-activating factor (PAF). Notably, mice that are double knockout for Src-1/-2 die at birth of respiratory distress, due to decreased surfactant lipoprotein production. Intriguingly, we observed that wild-type (WT) mothers carrying Src-1/-2d/d fetuses manifested a ~38 h delay in parturition compared to WT mothers carrying WT fetuses. This was associated with decreased production of SP-A and PAF by the Src-1/-2d/d fetal lungs. Our findings suggested that these effects of Src-1/-2d/d were caused by impaired glucocorticoid receptor (GR) transcriptional activity in fetal lung cells. To identify other genes in fetal lung that were affected by Src-1/-2d/d, we conducted RNA-seq analysis of lungs from 18.5 days post-coitum Src-1/-2d/dvs. WT fetuses. We observed that one of the genes most highly downregulated in Src-1/-2d/d fetal lungs was 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). 11β-HSD1 catalyzes the conversion of inactive cortisone or 11-dehydrocorticosterone into active cortisol or corticosterone, respectively, which are ligands for the glucocorticoid receptor (GR). We validated the RNA-seq results by RT-qPCR and immunoblotting and observed a striking reduction of 11β-HSD1 mRNA and protein in lungs of Src-1/2d/d fetuses, compared to WT. Others observed that glucocorticoids potently increased 11β-HSD1 expression in various cell types via activation of transcription factors C/EBPα and C/EBPβ, providing a potential positive feed-forward loop. Notably, we observed that C/EBPα and C/EBPβ mRNA and protein were markedly reduced in Src-1/-2d/d fetal lungs, compared to WT. Deletion of the Cebpa gene in respiratory epithelium of fetal mice caused respiratory failure at birth due to surfactant lipid and protein deficiency. This was associated with increased expression of TGF-β2, which inhibits fetal lung maturation. Notably, we observed that expression of TGF-β2 and TGF-β3 were increased in Src-1/-2d/d fetal lungs. Thus, impaired lung development, surfactant synthesis and delayed parturition in Src-1/-2d/d fetuses are likely caused by decreased 11β-HSD1 and GR signaling, resulting in decreased C/EBPα/β expression and increased TGF-β signaling.
The softgel, vaginal 17β-estradiol (E2) insert (TX-004HR) significantly improved the maturation index of the vaginal epithelium, dyspareunia and vaginal dryness in menopausal women with vulvar and vaginal atrophy (VVA) and moderate to severe dyspareunia, without histological changes to the endometrium (Constantine G et al, Menopause 2017;24:409-416). The 4-μg and 10-μg E2 doses were FDA approved as Imvexxy® (TherapeuticsMD, Boca Raton, FL), for the treatment of moderate to severe dyspareunia, a symptom of VVA, due to menopause. The progesterone receptor (PR) is an estrogen-regulated gene with expression that is very sensitive to E2 exposure (Xiao CW and Goff AK, J Reprod Fertil.1999;115:101-109). Endometrial PR expression in the biopsies of women using the softgel vaginal 4-µg E2 insert was used as a marker of E2 exposure to determine whether sufficient E2 applied with the vaginal insert reaches the endometrium to upregulate PR expression. Our hypothesis posits that there would be insufficient E2 from the 4-µg E2 insert to stimulate an increase in endometrial PR expression. In this post hoc analysis of the REJOICE trial, endometrial biopsies from 25 women who had a normal baseline biopsy, an on-therapy biopsy after study day 70, and tissue readings from all pathologists were randomly selected from the 4-µg E2 vaginal insert and placebo groups. Endometrial tissue sections were stained to visualize PR expression using PgR1294 (Agilent; Santa Clara, CA). Cell staining was quantified using a trainable feature-recognition algorithm and mean expression levels between baseline and week 12 were analyzed by 2-sided t-tests. Acceptable PR expression results were available for 22 women in the 4-µg E2 group (three had insufficient tissue for analysis) and 25 women in the placebo group. For the 4-µg E2 group, mean ± SD (pmol/mg) PR expression was 0.455 ± 0.203 at baseline and 0.506 ± 0.226 at week 12. For the placebo group, mean PR expression was 0.579 ± 0.196 at baseline and 0.563 ± 0.213 at week 12. Mean PR expression levels at baseline and week 12 were not significantly different from each other within the 4-µg E2 (P=0.438) or placebo (P=0.783) group. No meaningful difference in endometrial PR expression was observed with the vaginal 4-µg E2 insert at week 12. These data support our hypothesis and the assertion that low-dose, local vaginal E2 exposure with the insert placed in the lower part of the vagina, does not pose an endometrial safety issue in postmenopausal women.
Background: In 1987, Graves observed that during the 3rd trimester, some patients with pre-eclampsia had high levels of unknown materials that could be detected with assays for digoxin (DLM). In 2018, we characterized a new candidate for the DLM, Ionotropin. It is a phosphocholine (PC) ester of a novel steroid with 23 carbon atoms. As Ionotropin shares structural features (a) with spironolactone (both have spiral lactones in the E-ring) and (b) with digoxin (E-ring lactone and 3α-5β configuration), we have proposed that Ionotropin may function as a potassium (K+) sparing diuretic. This suggestion is supported by the observations that [1] patients who cannot make Ionotropin (7-dehydrosterol reductase deficiency) are K+ wasting and [2] breast cyst fluids with high K+ levels also have high Ionotropin levels. Hypothesis: During the 3rd trimester, fetal requirements for K+ reach a maximum, fetal blood pressure increases and aldosterone signaling is blocked. This blockage leads to fetal sodium (Na+) wasting and is essential for formation of amniotic fluid. These events are consistent with a normal role for an unknown endogenous K+ sparing hormone and would be the basis for a modest elevation of maternal DLM during the 3rd trimester. Our hypothesis is that if any of the functions were inadequate, then the fetal-placental unit would synthesize excess PC-spiral steroids; the woman would exhibit symptoms of K+ sparing hormone excess (hypertension and proteinuria) and would be diagnosed with pre-eclampsia. Experimental Results: We have just reported a pilot study associating elevated PC esters of spiral steroids in women with pre-eclampsia. In brief, 12 of 19 women had elevated levels of at least one of the PC steroids (Z-score > 2) when compared to the levels in 20 pregnant women matched for gestational age and fetal sex. There are two basic mechanisms for this dichotomy: (a) there may be episodic secretion with of a DLM with a short half-life or (b) there may be two different underlying biochemical causes. In prior studies, there has been no indication of episodic secretion of DLM similar to that observed with glucocorticoids, Ionotropin or other PC spiral steroids. Discussion: There are two basic types of K+ sparing diuretics. Type A: Spironolactone functions by regulating the NaK-ATPase. Type B: Triamterene functions by blocking synthesis of epithelial Na+ channels. Thus, Type A would have high levels of spiral steroids and Type B would have low levels of spiral steroids. Type A patients would be expected to have higher risk of long-term consequences when compared to the Type B patients. Conclusion: The recognition of the division of pre-eclampsia into two separate diseases might be the key observation for developing Type-specific diagnosis and therapy. For example, a Type A patient might benefit from a low salt diet but that diet would not be expected to benefit a patient with Type B disease.
Introduction: Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in people with diabetes globally. Based on recent cardiovascular outcomes trial (CVOT) data, antihyperglycemic therapies have the potential to reduce cardiovascular (CV) risk in patients with type 2 diabetes (T2D). We sought to assess the baseline knowledge and the effect of a live continuing medical education (CME) on internal medicine physicians regarding knowledge of recent CVOT data. Method: A live, interactive satellite symposium was held at the American College of Physicians (ACP) annual meeting. We assessed the effects of the symposium education using a pre-/post-assessment study design. During the symposium, participants were given iPads to answer pre/post questions and the post-activity evaluation. The pre-/post-assessment instrument included knowledge- based questions. Pearson’s chi-squared test assessed whether the post‐assessment score differed from the pre‐assessment score. P values are shown as a measure of significance; P values <.05 are considered statistically significant. Results: A total of 182 physicians attended the symposium, of which 135 (74%) participated in the interactive questions via iPad. Significant overall improvements were seen in all but one area. • There was a 22% positive difference from pre- to post-assessment in the percentage of participants who correctly identified the >10% reduction in fatal/nonfatal MI/stroke that a 1% reduction in A1c would correlate with (25%, n=105 pre; 47%, n=91 post; P=.001). • There was a 6% positive difference from pre- to post-assessment in the percentage of participants who recognized the 14% reduction in 3-point MACE demonstrated in the EMPA-REG CVOT trial (36%, n=98 pre; 45%, n=105 post-assessment; P=.242). • Finally, there was a 41% positive difference from pre- to post-assessment in the percentage of participants who recognized that both canagliflozin and empagliflozin had demonstrated a >30% reduction related to hospitalizations with heart failure line (25%, n=89 pre; 66%,n=100 post; (P<.001). Ongoing educational gaps identified by low post-assessment knowledge levels include: 53% of participants still failed to recognize the correlation between reduction in A1c and reduction of CV events, 55% of participants still failed to identify 3-point MACE reductions from a specific CVOT, and 34% still failed to identify similarities in CVOT related to HHF. Conclusion: This study demonstrates the success of a case-based, interactive live satellite symposium on improving knowledge of internal medicine physicians related to CVOT data. The ongoing educational gaps identified in this activity demonstrate a need for additional education focused on improving knowledge of data from CVOT. Further, internal medicine physicians could also benefit from deeper case-based education focusing on application of CVOT data.
Androgen analogy (Oxandrolone) have been reported to better maintain lean body mass, with improved hypermetabolic responses and shortened healing time for major burn injured patients. This is contradictory to that androgens inhibit local wound repair in men and male mice. The aim of this study therefore is to identify the role of pure androgen dihydrotesterone (DHT) in complex major burn injury, in particularly whether androgen targets local healing process or systemic burn induced hypermetabolism. A DHT silastic tube was subcutaneously implanted to male Balb/c mice prior to surgery as the treatment group. A 2 X 2 cm² full thickness contact burn wound was created on the dorsal skin of wild type littermates (control) or DHT treated mice. Wound healing rate and body weight changes were measured and compared between treatment and non-treatment group. The serum level of inflammatory cytokine/chemokine was measured using a Multiplex Immunoassay System. Spleen immune cells enumeration was analysis by flow cytometry. Inflammation, re-epithelialization, cell proliferation and collagen deposition was analysed using histology, immunohistochemistry and RT-PCR. In the present study, we found DHT treatment better maintained the body weight in mice and significantly promoted wound healing over 14 days, whereas DHT treatment had no effect on burn-induced hypermetabolism. In control group, major burn injury triggered an acute systematic inflammation response, resulting in significant increased weight of spleen, excess infiltration of nucleated erythroid cells in red pulps of spleen and a significant increase in number of splenic monocytes over 21 days. DHT treatment shortened the systemic inflammation response, evidenced via reduced splenic weight and the number of monocytes in spleen and circulation at day 14 and 21. This finding is further confirmed by less infiltration of macrophages in wound area at day 14 and 21 compared to control group. Taken together, our results suggesting the DHT treatment significantly improve wound healing by accelerated turnover of inflammation response but not through the metabolism. Further studies are necessary to define the exact mechanisms and DHT treatment could be a new therapeutic approach to improve the survivability of major burn injured patient.
Activating somatic mutations to the ligand binding domain (LBD) of ESR1 (the gene for estrogen receptor alpha) may arise after prolonged hormone treatments. Y537S and D538G are the most prevalent mutations, accounting for 16% and 32% of cases respectively. Both mutations resist tamoxifen by favoring the receptor’s active state in the absence of hormone to reduce the selective estrogen receptor modulator’s binding affinity. Fulvestrant, a selective estrogen receptor degrader, is the only clinically approved molecule that can fully ablate Y537S estrogen receptor alpha (ERα) transcriptional activity, but does so at decreased potency due to a reduced ligand binding affinity. Interestingly, lasofoxifene, a selective estrogen receptor modulator, possesses significant anti-tumor activity in preclinical models of luminal breast cancers harboring WT, Y537S, and D538G ERα. Comprehensive structural and biochemical studies were undertaken to determine the molecular basis for lasofoxifene potency towards Y537S ERα. X-ray crystal structures of lasofoxifene in complex with WT and Y537S ERα LBD show that the molecule adopts an identical conformation within the hormone binding pocket. While, a competitive ligand binding assay shows that lasofoxifene possesses the same affinity towards WT and Y537S ERα LBD in vitro, compared to 5-10 fold decrease compared to other SERMs and SERDs. Collectively, these data suggest that lasofoxifene achieves improved anti-tumor activity by retaining its high affinity binding for the Y537S ERα somatic mutant.
Introduction: It is well known that estrogen plays an important role in thyroid regulation. We report an unusual case of post-partum placenta accreta causing pathologic estrogen secretion leading to increased levothyroxine (LT4) requirements and inability to lactate. Case: A 36-year-old woman with history of Hashimoto’s hypothyroidism presented post-partum day 11 after a normal vaginal delivery with inability to produce breast milk and mildly elevated TSH levels. Prior to her pregnancy, she required an equivalent dose of 142 mcg of LT4 supplementation daily, which increased appropriately to 171 mcg during pregnancy. After delivery, LT4 was decreased to 150mcg in anticipation of normalization of levothyroxine requirements to pre-pregnancy level. However, she had difficulty lactating and was found to have elevated prolactin, estradiol, and TSH levels. The following day, she presented to her obstetrician for persistent vaginal bleeding and was found to have placenta accreta requiring dilation and curettage (D&C). Her LT4 requirements eventually dropped to 125 mcg with decreasing beta-HCG and estrogen levels after successful D&C treatment. She was also then able to produce sufficient breast milk for lactation. Discussion: This case highlights the effect of estrogen on LT4 requirements during physiologic pregnancy and postpartum with placenta accreta. It is expected that hypothyroid patients have approximately 25-50% increased thyroid replacement requirements during pregnancy, which normalizes soon after delivery.1 Estrogen increases thyroxine-binding globulin and lowers circulating free thyroxine2,, which causes higher thyroid replacement requirements. Estrogen is also known to inhibit lactation. Our patient demonstrates that this holds true even in a pathologically high estrogen state from placenta accreta. Our case uniquely demonstrates a temporal association between estrogen levels and LT4 requirements in the post-partum hypothyroid patient. Patients with inappropriately high TSH levels after delivery should prompt investigation into pathologic causes of elevated estrogen-states, as levothyroxine requirements are expected to normalize immediately post-partum. References: 1. Bungard TJ, Hurlburt M. Management of hypothyroidism during pregnancy. CMAJ. 2007;176(8):1077-8. 2. Alexander EK, Marqusee E, Lawrence J, Jarolim P, Fischer GA, Larsen PR. Timing and magnitude of increases in levothyroxine requirements during pregnancy in women with hypothyroidism. N Engl J Med. 2004 Jul 15;351(3):241-9.
Background: Despite improvements in available options for the management of hypoparathyroidism and its significant impact on quality of life, this disorder remains suboptimally managed. We sought to determine if an online, virtual patient simulation (VPS)-based continuing medical education (CME) intervention could improve performance of diabetologists/endocrinologists (D/Es) and primary care physicians (PCPs) in the evidence-based management of hypoparathyroidism. Methods: The intervention comprised two patients presenting in a VPS platform that allows learners to order lab tests, make diagnoses, and prescribe treatments supported by an extensive database of diagnostic and treatment possibilities matching the scope and depth of actual practice. Tailored clinical guidance (CG), based on current evidence and expert recommendation, was provided following each decision, accompanied by the opportunity for the learner to modify to their decisions. Decisions were collected post-CG and compared with each user’s baseline (pre-CG) decisions using a 2-tailed paired t-test to determine P values. Absolute percent difference was calculated and is presented as percent improvement in the results. Results: Significant improvements were observed after CG: Case 1 (n=148 D/Es, n=566 PCPs): • Appropriate diagnosis of hypoparathyroidism: 26% improvement among D/Es (47% pre-CG vs 73% post-CG; P<.001), 27% improvement among PCPs (42% pre-CG vs 69% post-CG; P<.001) o Further, most appropriate diagnosis of hypoparathyroidism status post-subtotal thyroidectomy: 45% improvement among D/Es (17% pre-CG vs 62% post-CG), 49% improvement among PCPs (7% pre-CG vs 56% post-CG; P<.001) • Order patient education: 22% improvement among D/Es (44% pre-CG vs 66% post-CG; P<.001), 18% improvement among PCPs (51% pre-CG vs 69% post-CG; P<.001) Case 2 (n=92 D/Es, n= 381 PCPs): • Initiate parathyroid hormone: 37% improvement among D/Es (14% pre-CG vs 51% post-CG; P<.001), 41% improvement among PCPs (11% pre-CG vs 52% post-CG; P<.001) • Order patient education: 16% improvement among D/Es (38% pre-CG vs 54% post-CG; P<.001), 19% improvement among PCPs (42% pre-CG vs 61% post-CG; P=.012) Conclusion: VPS that immerses and engages specialists in an authentic and practical learning experience can improve evidence-based clinical decisions related to management of hypoparathyroidism.
Triple negative breast cancer (TNBC) patients have a worse prognosis relative to other breast cancer subtypes. The glucocorticoid receptor (GR) is a ubiquitous steroid receptor that mediates homeostatic responses to life-induced stressors via glucocorticoids synthesized in the adrenal cortex. High GR expression predicts poor outcome in TNBC. The molecular mechanisms for how stress and GR contribute to TNBC progression are largely unknown. We previously described overexpression of protein tyrosine kinase 6 (PTK6) in response to both cellular and endocrine stress, coordinated by GR/HIF complexes in TNBC models. PTK6-induced signaling further amplified phosphorylation of GR on Ser134. PTK6 knock-down impaired TNBC motility, and blocked outgrowth of cancer stem-like cells (CSC) as measured by tumorsphere assays. Furthermore, PTK6 expression was integral for cancer cell survival upon chemotherapy treatment (Taxol, 5-FU). Interestingly, kinase activity of PTK6 was not required for TNBC cell motility or CSC outgrowth. This finding is of critical importance for drug design, given that previous efforts have primarily focused on targeting the PTK6 kinase domain. To probe the mechanisms of PTK6 signaling, we have created kinase-intact domain structure mutants of PTK6 via in frame deletions of the N-terminal SH3 or SH2 domain. MDA-MB-231 cells expressing PTK6 which lacks an SH2 domain (SH2-del PTK6) exhibited increased formation of tumorspheres relative to wt or kinase-dead (KM) full-length PTK6. Surprisingly, SH2-del PTK6+ cells were strikingly less responsive to both serum- and growth factor-stimulated cell motility relative to wt or KM controls. To track signal transduction pathways activated in TNBC cells harboring PTK6 domain mutants (wt, KM, SH2-del PTK6, PTK6 null), we used Reverse Phase Protein Array (RPPA). Our data suggests that the SH2 domain of PTK6 mediates TNBC motility via activation of the Rho and/or AhR signaling pathways, while CSC outgrowth primarily occurs via activation of the p38 and/or canonical Wnt signaling pathways. Together, these studies nominate PTK6 as an effector of advanced cancer phenotypes in GR+ TNBC cells and identify novel therapeutic targets (Rho, AhR) for treatment of TNBC tumors. Additionally, our work defines PTK6 domain-specific functions and reveals new opportunities for pharmacologically targeting the PTK6 SH2-domain in TNBC.
Gas5 is a long non-coding RNA initially identified as a factor inducing cell growth arrest and apoptosis. Several stem-loop structures in Gas5 3’-terminal sequence mimic hormone response elements (HRE) with different binding affinities towards estrogen, glucocorticoid and mineralocorticoid receptors. Based on this property, Gas5 acts as a decoy for steroid nuclear receptors, competing with genomic HREs and downregulating receptor activity. In this study we asked whether Gas5 is an aldosterone-regulated gene in vivo and studied its effect on mineralocorticoid and glucocorticoid receptor activity (MR and GR). Our results show that Gas5 is expressed in the rat distal colon epithelium and its abundance increased after four days of low Na+ diet in an MR-dependent fashion. In addition, MR transfected in HEK293 cells regulates Gas5 expression in response both to aldosterone and cortisol. Co-expression of Gas5 with either MR or GR in COS-7 cells confirmed previous findings showing that it decreases receptor-mediated transactivation. However, when both MR and GR where co-expressed, Gas5 reduced cortisol- but not aldosterone-dependent reporter gene transactivation. This effect does not depend on alterations of receptor expression, nuclear translocation or dimerization, but can be ascribed to differential affinities in MR and GR binding to Gas5. Our results suggest a role for Gas5 selectively regulating corticosteroid signaling in the rat colon.
Background: We have previously examined the link between diabetes mellitus (DM) and malignancy with respect to glycemic control and survival. Given that mortality data derived from our own records is limited, and to better determine if DM changes mortality risk in cancer patients with co-existing DM, we linked our clinical records with the National Death Index (NDI), housed and maintained by the Centers for Disease Control and Prevention. Objective: The primary objective of this study was to evaluate the risk DM imposes on mortality in cancer patients via the National Death Index. The secondary objective was to ascertain the cause of death in cancer patients with and without DM. Methods: We identified 1565 patients with newly diagnosed prostate, breast, lung, colorectal, and pancreatic cancer from the institutional cancer registry from 2010-2015. This data was forwarded to the CDC/NDI for input on whether each patient was alive or deceased, and cause of death (if applicable). NDI records were reviewed and verified with a resulting data set containing 1404 patients, matched 2:1; cancer without DM (n=936) and cancer and concurrent DM patients (n=468). Patients were matched by year of diagnosis, age, gender, and cancer type. Primary cause of death was categorized into cancer-related, vascular, non-cancer/non-vascular, and unknown. Five-year survival was estimated with the Kaplan-Meier method and Cox proportional hazards regression analysis was used with the matched pair as the strata variable. Results: Distribution of cancer types was: prostate (43%), lung (19%), breast (15%), colorectal (12%) and pancreatic (11%). Mean (SD) age at diagnosis was 68 (10) years; 70% of patients were male. There were 435 deaths with 168 deaths (36%) in the DM cohort and 267 (29%) in the non-DM cohort. Median follow-up was 47.3 (0.5-94.1) months across all cancer types. Overall 5-year survival was estimated at 59% (95% CI: 54-64%) for DM patients versus 67% (95% CI: 64-71%) in non-DM patients. Matched pairs Cox regression was HR= 1.35; 95% CI 1.02-1.79; p =0.04. There were 134 cancer related deaths (79%) in the DM cohort, and 233 (87%) in the non-DM cohort. There were 26 non-cancer, non-vascular deaths (15% in the DM cohort, and 13 (5%) in the non-DM cohort (p<0.001). Conclusions: Overall there is an increased risk of death for DM vs non-DM patients who have cancer. There is a higher proportion of non-cancer, non-vascular deaths in DM patients than in non-DM patients.
WT vs. S134A-GR transcriptomes in TGFβ1-treated MDA-MB-231 cells. a Principal component analysis plot derived from RNA-seq shows the differences in transcriptomes between MDA-MB-231 cells expressing either WT or S134A-GR cells and treated with vehicle or 10 ng/mL TGFβ1 for 6 h. b Volcano plot showing differential expression of genes in wt-GR+ compared to S134A-GR+ TNBC cells treated for 6 h with either Vehicle (left) or 10 ng/mL of TGFβ1 (middle). Volcano plot using the generalized linear model approach in EdgeR (comparisons of wt-GR and S134A-GR in response to TGF) to illustrate the effects of TGFβ1 treatment of cells expressing wt-GR relative to cells expressing S134A-GR (right). Red dots indicate genes with an absolute log2 fold-change of 1.0 or more and a false discovery rate of less than 0.05. Genes for GLM are detailed in Supplementary Table 1A. c Supervised heatmap shows significant differences in gene expression as measured by RNA-seq in TGFβ1-treated MDA-MB-231 cells expressing either wt-GR or S134A-GR. Rectangle indicates a cluster of interest in which genes are upregulated in response to TGFβ1 in wt-GR cells but not in the S134A-GR cells. All genes that were used for differential expression analysis are represented in this heatmap (n = 19,760). d DUSP1 and SGK1 mRNA levels were assessed using qRT-PCR following normalization to 18S expression. Mean expression of three independent replicates ± SD is shown. e Cell function and mechanism analyses of pathways via IPA of differentially expressed genes with an absolute log fold-change of 1.5 or more and a p-adjusted value of less than 0.05 in wt-GR relative to S134A-GR TNBC cells treated for 6 h with either Vehicle or TGFβ1 (10 ng/mL). f Upstream regulator analyses via IPA of differentially expressed genes with an absolute log fold-change of 1.5 or more and a p-adjusted value of less than 0.05 in wt-GR relative to S134A-GR TNBC cells treated with either Vehicle or 10 ng/mL of TGFβ1. Activation/inhibition z-scores are shown as well. Red dotted line indicates the significance value of 1.3 (p < .05)
Triple negative breast cancer (TNBC) is the most metastatic and deadliest breast cancer (BC) subtype, accounting for 20-30% of all BCs. There is a critical need to identify molecular targets that could be exploited as new biomarkers of TNBC prognosis and for improving therapies. Although TNBC lacks estrogen and progesterone receptors, 15-40% of TNBC patients express the glucocorticoid receptor (GR). Women with TNBC that express high levels of GR have poor outcomes. We hypothesize that GR is a key mediator of advanced cancer phenotypes in TNBC. Specifically, we propose that GR acts as a “sensor” for stress signaling pathways commonly activated by soluble factors that are abundant within the tumor microenvironment (TME). Using TNBC models, we showed previously that GR is phosphorylated on Ser134 in response to cellular stress stimuli such as hypoxia. Herein, we show that pS134-GR is elevated in TNBC tumor tissue samples relative to non-TNBC tissues. In vitro studies in TNBC models demonstrate that GR Ser134 phosphorylation is promoted by cytokines (TGFbeta), and growth factors (HGF) and occurs in the absence of GR ligands such as Dexamethasone or cortisol. In response to stress signaling inputs, studies with kinase inhibitors confirmed that p38 MAPK is required for GR Ser134 (pS134-GR) phosphorylation. To evaluate the functional significance of pS134-GR, we created CRISPR models of MDA-MB-231 TNBC cells expressing either wt GR or phospho-mutant GR that cannot be phosphorylated at Ser134 (S134A). RNAseq studies were performed to identify pS134-GR target genes in TNBC models. Our transcriptome data demonstrated a requirement for pS134 GR in the expression of gene sets associated with TGFβ and p38 MAPK signaling. Pathway analysis revealed that pS134-GR target genes primarily regulate cancer cell migration. In vitro assays revealed that pS134-GR is essential for inducing cell migration and anchorage-independent growth in TNBC cells, even in the absence of exogenous GR ligands. Furthermore, using co-IP assays, we identified that upon phosphorylation at Ser134, GR interacts with the scaffolding protein 14-3-3zeta. Like pS134-GR, 14-3-3zeta is highly expressed in TNBC when compared to non-TNBC patients. We observed co-recruitment of both pS134-GR and 14-3-3zeta to known pS134-GR target genes (i.e. PTK6) in TNBC cells. These data prompted us to test the requirement for 14-3-3zeta in GR-mediated phenotypes. Short hairpin RNA knock-down experiments demonstrated that expression of 14-3-3zeta is required for serum and TGFbeta-induced TNBC cell migration. We conclude that the pS134-GR/14-3-3zeta complex is a key “sensor” of local stress signals within the TME (TGFbeta) and a potent mediator of cell migration in TNBC models. Further studies are aimed at exploring pS134 GR as a biomarker and therapeutic target in TNBC.
Glucocorticoids are prescribed in many chemotherapy protocols for neoplasms of lymphoid origin based on their ability to inhibit lymphocyte proliferation and promote cell death (apoptosis). Lymphocytes have been shown to be profoundly sensitive to glucocorticoid-induced apoptosis, however glucocorticoid resistance restricts the efficacy of these stress hormones and reduces their clinical value. Bcl-2 is an anti-apoptotic protein that promotes cell survival and resistance to cell death during glucocorticoid therapy. Predictably, Bcl-2, as well as other anti-apoptotic Bcl-2 family members, have been found to be overexpressed in a variety of human cancers, including in over 90% of human follicular lymphomas and 95% of chronic lymphocytic leukemias. Unfortunately, approaches to overcome Bcl-2 resistance in cancer cells including anti-sense oligonucleotides, drugs that inhibit Bcl-2 function, and BH3 mimics have not been effective. We took a unique approach to overcome Bcl-2 resistance in tumor cells expressing this anti-apoptotic protein by first identifying points along glucocorticoid-induced cell death signaling program that are explicitly inhibited by Bcl-2. We then intervened at this stage that bypasses the point of inhibition. Provocatively, drugs such as CCCP and several microbial toxins which disrupt the mitochondrial membrane potential, when used in combination with glucocorticoids overcome the resistance afforded by Bcl-2 and kills the cells. Our data suggests drugs which alter the mitochondrial membrane potential in combination with glucocorticoids provide an effective approach in killing lymphocytic tumor cells expressing Bcl-2. This combination should be considered in accompanying current chemotherapeutic drug combinations to provide additional therapeutic benefit in overcoming Bcl-2 resistant tumors.
Purpose: The etiology of functional hypothalamic amenorrhea (FHA) can involve both metabolic and psychogenic stressors. The role of metabolic stress has been described in exercising women who develop FHA secondary to chronic energy deficiency attributable to inadequate dietary intake in the face of exercise training. The potential for psychological factors to contribute to exercise-related FHA is unknown. Methods: In our cross-sectional comparison of exercising women ((n=61), exercise ≥2 hours/week, age 18-35 years, BMI 16-25 kg/m2, we tested whether psychological factors discriminated participants who were deemed eumenorrheic or had FHA as confirmed by physical examination, health history, metabolic and endocrine screening, menstrual calendars and daily urinary collection for reproductive hormone metabolites. Body composition, energy balance, and metabolic and endocrine parameters were assessed in addition to psychological factors (Dysfunctional Attitudes Scale, Brief-Resilient Coping, Daily Stress Inventory, Perceived Stress Scale (PSS), Profile of Mood States (POMS), Beck Depression Inventory) and eating behaviors (Eating Disorder Inventory-2, Three-Factor Eating Questionnaire). Results: Exercising women with FHA had a significantly lower BMI (20.2 vs 21.5 m/kg2, p<0.05), less body fat (22.8 vs 25.8%, p<0.05), and a lower fat mass (56.2 vs 58.3 kg, p<0.05), compared to eumenorrheic women. Fasting total triiodothyronine (75.4 vs 89.8 ng/dL, p<0.001), leptin (5.2 vs 9.0 ng/dL, p<0.01), and the ratio of actual to predicted resting metabolic rate (0.84 vs 0.92, p<0.01) were significantly lower in FHA women. FHA women demonstrated a greater drive for thinness (2.9 vs 2.1, p<0.05), greater dietary cognitive restraint (11.3 vs 7.4, p<0.001), and displayed more dysfunctional attitudes, i.e., need for social approval (39.1 vs 33.7, p<0.05) compared to eumenorrheic women but there were no differences in perceived stress, depression, mood states, brief resilient coping, or daily stress (p >0.05). Notably, FHA women displayed a significant positive correlation between need for social approval and indicators of stress (PSS: r=0.50), depression (Beck: r=0.59), and mood (POMS-depression/dejection: r=0.55), which was not apparent in eumenorrheic women. Additionally, EDI-drive for thinness was significantly positively correlated with many of the variables associated with stress (PSS: r=0.47), depression (Beck: r=0.51), dysfunctional attitudes (r=0.55), and mood disorders (POMS-depression-dejection: r= 0.37; tension/anxiety: r=0.44)(all p<0.05). Conclusion: In exercising women, psychological factors do not overtly discriminate reproductive status; however, in women with FHA, there appears to be a higher need for social approval and restrictive eating behaviors may be related to underlying indicators of psychological stress and depression. US DoD PR054531
BACKGROUND: Endometriosis is an estrogen-dependent, inflammatory disease, and the role of estrogen is obvious because the symptoms associated with endometriosis often disappear after menopause, and GnRH agonists or progestin relieve the pelvic lesions and endometriosis-associated pain. However, there are limitations to these treatments that target the estrogen reduction in endometriotic lesions. We sought to define an aberrant gene expression derived from an epigenetic background in endometriosis.Objective: In the hope of overcoming the limitations of endocrine treatments in endometriosis, we examined estrogen receptor (ER)-dependent and -independent gene expressions promoted by active enhancers specifically hypomethylated in endometriotic cells. Patients: Institutional Review Boards approved this project. We obtained the informed consent from all patients. The chocolate cyst lining in ovaries of patients with endometriosis was the source of endometriotic tissue. As the control, the eutopic endometrial tissues were obtained from uteri of premenopausal women who had uterine leiomyoma.Methods: Stromal cells were prepared from endometriotic and endometrial tissues. Gene expression was examined using RT-PCR. The potential function of hypomethylated gene sequence as an active enhancer was evaluated by ChIP analysis using anti-H3K4me1 and anti-H3K27ac antibodies and eRNA expression analysis. Using ChIP-seq and ChIA-PET analysis in silico, ER-specific loci within gene bodies and the up- and downstream regions were extracted. ER-dependent gene expression was examined using estradiol or SERM.Results: ER expression in endometriotic cells.1) Relative expression of ERα mRNA was estimated to be one tenth of that in endometrial cells. 2) Relative expression of ERβ1 mRNA was 40-fold higher than that in endometrial cells, which is at a comparable level of the ERα. 3) ERβ2 mRNA expression was at a comparable level of the ERβ1. From our DNA methylation and gene expression analysis, 6 genes were selected and classified into 3 categories: estrogen-responsive genes with specific methylation (ESR1 and ESR2) or without any methylation (TGFα and GREB1), and estrogen-unresponsive but upregulated genes depending on specific hypomethylation (GATA6 and CYP19). 4) ChIP-seq and ChIA-PET analysis in silico suggested the presence of ER-specific loci within gene bodies and the up- and downstream in estrogen-responsive genes. 5) ChIP and eRNA expression analysis predicted active enhancer regions both in estrogen-responsive and -unresponsive genes. 6) In response to estrogen, TGFα and GREB1 expressions were upregulated, but ESR1 and ESR2 showed marginal responses.Conclusion: We focused on estrogen-responsive and -unresponsive genes linked to the epigenetic environment of endometriotic lesions, and revealed a facet of gene expression in endometriotic cells.
Anti-Müllerian hormone (AMH), an inhibitor of primordial/small antral follicle development and Leydig cell androgen synthesis in mice, could exaggerate the polycystic ovary syndrome (PCOS) phenotype, given reports of PCOS-specific AMH loss-of-function mutations (1–3). This report describes a normal-weight PCOS woman with severely reduced AMH levels (index PCOS woman). It examines the molecular basis for her reduced serum AMH levels and also compares her endocrine characteristics to similar-weight PCOS women with detectable AMH. Twenty normo-androgenic ovulatory (control) and 13 age- and body mass index-matched PCOS women (19–35 years; 19–25 kg/m2) underwent transvaginal sonography and serum hormone measures. Wilcoxon rank-sum test compared clinical features of control and PCOS women with detectable AMH, which were then individually ranked by magnitude in all PCOS women. DNA analysis was performed by PCR amplification with direct gene sequencing. The identified mutation was introduced in hAMH-expression plasmids for functional analysis of AMH processing in HEK293 cells by Western blot and ELISA (pico-AMH assay, Ansh Labs, Webster, TX), and for bioactivity in KK-1/AMHR2 cells using a luciferase reporter. Unpaired t-test compared AMH-induced luciferase activity between wild type and mutant AMH. A homozygous AMH gene mutation rs10417628 involving a single base pair substitution in exon 5 (NG_012190.1:g.7705C>T, p.(Ala515Val)) was identified in the index PCOS woman. PCOS women with detectable AMH had higher serum AMH (10.82 [6.74–13.40] ng/mL, Median [IQR]), total/free testosterone (T) (total T: 55.5 [49.5–62.5] ng/dL; fT: 5.65 [4.75–6.6] pg/mL) levels and greater total antral follicle numbers (AFNs) (46 [39–59] follicles) than controls (AMH: 4.03 [2.47–6.11] ng/ml; total T: 30 [24.5–34.5] ng/dL; fT: 2.2 [1.8–2.45] pg/mL; AFNs 16 [14.5–21.5] follicles, P<0.05, all values), along with a trend toward LH hypersecretion (P=0.06). The index PCOS woman with the lowest AMH levels (0.1 ng/ml) did not have the highest serum total T/fT (total T: 89 ng/dL; fT: 7 pg/mL,) or LH levels nor the greatest AFN (43 follicles). In vitro analysis of cells expressing hAMH-515Val or hAMH-515Ala showed that hAMH515-Val, in contrast to hAMH515-Ala, was undetectable and severely reduced in the pico-AMH assay in cell lysates and supernatants, respectively. AMH protein processing and AMH-induced luciferase activity, however, did not differ between hAMH515Val and hAMH515Ala. Thus, homozygous AMH mutation rs10417628 in a PCOS woman can impair serum AMH immunoreactivity without affecting AMH bioactivity, perhaps because of conformational changes from the mutation that only interfere with its immunodetection but not its function. References: 1. Teixeira J, et al. Endocrinology 1999;140:4732 2. Gorsic LK et al. JCEM 2019;104:2855 3. Broekmans FJ, et al. Trends Endocrinol Metab 2008;19:340
Background: Gestational trophoblastic disease (GTD) represents a group of tumours caused by abnormal proliferation of trophoblastic cells, including molar pregnancy. Elevated β-hCG levels are an established marker for the presence of the disease and useful for monitoring. Due to the shared structural homology of β-hCG and TSH, hyperthyroidism can occur. Clinical Cases: We present two patients with GTD associated with hyperthyroidisim. Case 1, a 20 year old female (G1P0) presented to the emergency department complaining of vaginal bleeding associated with abdominal pain. She was estimated to be 13 weeks. Laboratory evaluation were β-hCG 648 324 IU/L, TSH 0.06 (0.35 - 4.94 mIU/L, free T4 23.2 (9.0 - 19.0 pmol/L, Hb 8.0 (11.6 - 16.4 g/dL). Ultrasound revealed molar pregnancy. She underwent uterine evacuation, thereafter complicated with thyroid storm (Burch Wartofsky score = 45). Post- operative vitals were BP 192/112, pulse rate 120 bpm and temperature 360C. She was managed in high care on labetolol, carbimazole, lugol’s iodine and hydrocortisone. She was subsequently referred to Medical Oncology for further management. Histology sample obtained in theatre confirmed complete molar pregnancy. Her staging CT scan indicated the presence of small lung nodules, suggesting metastatic disease. The patient’s FIGO/WHO score was III: 2. At the time of preparing this study, she had already received 7 weeks of methotrexate intramuscularly and still had detectable β-hCG levels. Case 2 was a 31 year old female presented similarly. This was her second pregnancy (G2P1), 12 weeks by dates. Her vitals were BP 141/74, pulse rate 110 bpm and temperature 36oC. The Ultrasound revealed larger for gestational age uterus with cystic structures in utero. Her quantitative β-hCG was significantly elevated (> 1 500 000 IU/L) she was thyrotoxic [TSH (<0.1 (0.34 - 4.94 mIU/L) free T4 (47.2 (9.0 - 19.0 pmol/L)], however did not develop thyroid storm (Burch Wartofsky score = 20). This patient also underwent uterine evacuation and did well post operatively. She was treated for her thyrotoxicosis with carbimazole, propranolol and thiamine. Further management was by Medical Oncology. Histological examination was in keeping with a partial mole. Her staging CT scan showed no metastasis, and had a FIGO/WHO score of 1: 4 due to her pre-treatment hCG of >1.5 million IU/L. She received 7 cycles of intramuscular methotrexate from which she achieved and maintained suppressed β-hCG levels (<1 IU/L). Conclusions: This study has demonstrated that the β-hCG levels may not always correlate with disease severity and prognosis. When comparing the two patients Case 1 had lower levels of β-hCG and of free T4 than Case 2, however was clinically more unwell, developed thyroid storm and had metastatic disease. Case 2 had hCG levels almost double those of Case 1, wsa stable and her levels decreased much quicker reaching undetectable levels
Congenital adrenal hyperplasia (CAH) is associated with poor health outcomes. This is, in part, because doses of glucocorticoid which are sufficient to suppress excess adrenal androgens are also associated with adverse metabolic effects such as insulin resistance. This toxicity occurs with efficacious doses of all commonly prescribed glucocorticoids (hydrocortisone, prednisolone and dexamethasone). However, the glucocorticoid corticosterone may have an improved therapeutic index because of its unusual susceptibility to export from cells by ATP-binding cassette (ABC) transporters. ABCB1 is expressed in the brain and exports cortisol (hydrocortisone), prednisolone and dexamethasone, limiting their potency at suppressing ACTH. However, corticosterone is not exported by ABCB1 but is exported by the alternative ABCC1 transporter. Expression of ABCC1 is relatively low compared to ABCB1 in the brain, however ABCC1 is expressed in the absence of ABCB1 in adipose tissue, muscle and bone, potentially limiting corticosterone action in these tissues. We hypothesized that corticosterone may be more efficacious at suppressing ACTH and adrenal androgens but with less metabolic toxicity than hydrocortisone. Fourteen adults with classic CAH due to 21-hydroxylase deficiency were recruited to a double-blind randomised crossover study comparing intravenous infusions of placebo, hydrocortisone and deuterated (D8) corticosterone. Subjects attended after omitting their usual glucocorticoid for 12h and were administered glucocorticoid/placebo for 5.5 hours in a two-step infusion designed to achieve concentrations of 400 and 800nM. Blood samples were collected regularly. Circulating D8-corticosterone concentrations were approximately 30% higher than hydrocortisone. D8-corticosterone suppressed ACTH, androstenedione and 17-hydroxyprogesterone to a greater extent than hydrocortisone. However, hydrocortisone increased circulating insulin compared with D8-corticosterone and placebo (10.0±1.3 vs 8.3±1.2 vs 7.2±1.3mU/L respectively, P<0.05). Blood pressure and FFAs were similar between phases. Thus, corticosterone acutely suppresses ACTH and adrenal androgens in CAH patients without causing hyperinsulinaemia. Corticosterone may be a better glucocorticoid replacement than hydrocortisone for the treatment of CAH.
Maturity onset diabetes of the young (MODY) is an autosomal dominant form of monogenic diabetes that is often misdiagnosed as type 1 diabetes (T1D) or type 2 diabetes (T2D). The most common MODY types are due to heterozygous mutations in the HNF1A, HNF4A, or GCK genes. Sulfonylureas are the first line treatment for HNF1A-/HNF4A-MODY, with stable or improved glycemic control compared to insulin (1). GCK-MODY, which manifests as stable hyperglycemia with no significant long-term complications, requires no treatment (2). Although early diagnosis leads to significant economic and quality of life benefits for patients with MODY, past cost-effectiveness studies have found that population-wide MODY genetic testing is not cost-effective due in part to low MODY prevalence (3). We sought to assess the combined effect of 1) biomarker screening to target genetic testing towards patients at high risk for MODY and 2) the addition of cascade genetic testing for first-degree relatives of affected probands on the cost-effectiveness of MODY genetic testing. We used simulation models of distinct forms of diabetes (HNF1A/HNF4, GCK, T1D, T2D) to forecast the clinical and economic consequences of this genetic testing strategy in pediatric patients over a 30-year horizon. In the testing arm, patients with HNF1A-/HNF4A-MODY switched to sulfonylureas, which was associated with a 1.0% reduction in HbA1c leading to lower rates of micro- and macrovascular complications. GCK-MODY patients switched to no treatment, without change in HbA1c. Patients with T1D or T2D experienced no change in care or outcomes in the testing arm. Study outcomes included costs, quality-adjusted life years (QALY), and the incremental cost-effectiveness ratio (ICER) (USD/QALY). Cost-effectiveness was defined as ICER less than $50,000 (typical threshold). Applying biomarker screening increased average quality of life (+0.0057 QALY) but also increased costs (+$280) in the testing arm relative to the control arm, yielding an ICER of $49,365. Adding cascade genetic testing increased quality of life (+0.0084 QALY) and lowered costs (-$268). Thus, applying biomarker screening to the MODY genetic testing strategy was cost-effective, and cascade genetic testing made the strategy cost-saving. National implementation of this strategy could improve the lives of patients with MODY while saving society money, which is a rare feat in personalized medicine. Reference: (1) Fajans and Brown, Diabetes Care, 1993;16(9):1254-1261. (2) Velho et al., Diabetologia, 1997;40(2):217-224. (3) Naylor et al., Diabetes Care, 2014;37(1):202-209. Sources of Research Support: Institutional Grant; The National Institute of Diabetes and Digestive and Kidney Diseases (P30, K24, K23, and R01)
During lactation, a woman experiences a considerable amount of bone loss and recent studies suggest bone deficits persist years postpartum. Furthermore, selective serotonin uptake inhibitors (SSRIs), which are often prescribed to women experiencing peripartum depression, have been linked to osteopenia. Serotonin signaling can increase parathyroid hormone related protein (PTHrP), a bone remodeling protein which liberates calcium for the milk. Additionally, fluoxetine (a common SSRI) results in increased mammary gland serotonin content and PTHrP, and treatment during the peripartal period reduced maternal bone mineral density. One proposed mechanism of serotonin action is by its covalent addition to proteins by transglutaminase (TG2), termed serotonylation. We therefore investigated whether the combination of fluoxetine and lactation can exacerbate maternal bone loss and the underlying mechanism. We hypothesized that SSRI-induced serotonin signaling in the lactating mammary gland increases PTHrP through a serotonylation-dependent mechanism. Treatment of mouse mammary epithelial cells (HC11) with fluoxetine significantly upregulates PTHrP gene expression and the concentration of its downstream effector, cAMP, over control (P < 0.0004). Furthermore, treatment of the HC11 cells with fluoxetine in addition to a TG2 inhibitor, monodansylcadaverine, restores PTHrP mRNA expression to levels observed in the control. Small g-proteins have emerged as a common target protein for serotonylation. Currently, our data suggest that the g-proteins, RhoA and Rab4, are potential serotonylation targets in the mammary gland. Together these data suggest that the molecular process of serotonyation in HC11 cells links serotonin signaling to increased PTHrP expression. Future work is directed at using the cre-lox system to genetically ablate serotonylation using a WAPCre/TG2Flox transgenic mouse to determine whether decreasing serotonylation in vivo in the mammary gland during lactation improves maternal bone mass.
Top-cited authors
Sharon E Oberfield
  • Columbia University
Selma Feldman Witchel
  • Childrens Hospital of Pittsburgh
Shobha Ghosh
  • Virginia Commonwealth University
Siddhartha S Ghosh
  • Virginia Commonwealth University
Grazia Aleppo
  • Northwestern University